Methods for allosteric modulation of the GABA receptor by members of the androstane and pregnane series

ABSTRACT

Methods, compositions, and compounds for modulating the GABA A  receptor-chloride ionophore complex to alleviate stress, anxiety, seizures, mood disorders, PMS and PND and to induce anesthesia.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.09/349,902, filed Jul. 8, 1999, now U.S. Pat. No. 6,143,736, which is adivisional of U.S. application Ser. No. 08/887,229, filed Jul. 2, 1997,now U.S. Pat. No. 5,939,545, which is a continuation of U.S. applicationSer. No. 08/389,820, filed Feb. 14, 1995, now abandoned, which is acontinuation-in-part of U.S. application Ser. No. 08/346,927, filed Nov.23, 1994, now abandoned, which is a continuation-in-part of U.S.application Ser. No. 08/196,919, filed Feb. 14, 1994, now abandoned, thecontents of each of which are fully incorporated by reference herein.

BACKGROUND OF THE INVENTION

The present invention is directed to methods, compositions, andcompounds for modulating animal (and human) brain excitability via thegamma-aminobutyric acid A (GABA_(A)) receptor-chloride ionophore complex(GRC). Specifically, the present invention is directed to methods,compositions, and compounds for modulating brain excitability throughbinding to the neurosteroid receptor site on the GRC.

Brain excitability is defined as the level of arousal of an animal, acontinuum that ranges from coma to convulsions, and is regulated byvarious neurotransmitters. In general, neurotransmitters are responsiblefor regulating the conductance of ions across neuronal membranes. Atrest, the neuronal membrane possesses a potential (or membrane voltage)of approximately −80 mV, the cell interior being negative with respectto the cell exterior. The potential (voltage) is the result of ion (K⁺,Na⁺, Cl⁻, organic anions) balance across the neuronal semipermeablemembrane. Neurotransmitters are stored in presynaptic vesicles and arereleased under the influence of neuronal action potentials. Whenreleased into the synaptic cleft, an excitatory chemical transmittersuch as acetylcholine will cause membrane depolarization (change ofpotential from −80 mV to −50 mV). This effect is mediated topostsynaptic nicotinic receptors which are stimulated by acetylcholineto increase membrane permeability to Na⁺ ions. The reduced membranepotential stimulates neuronal excitability in the form of a postsynapticaction potential.

In the case of the GRC, the effect on brain excitability is mediated byGABA, a neurotransmitter. GABA has a profound influence on overall brainexcitability because up to 40% of the neurons in the brain utilize GABAas a neurotransmitter. GABA regulates the excitability of individualneurons by regulating the conductance of chloride ions across theneuronal membrane. GABA interacts with its recognition site on the GRCto facilitate the flow of chloride ions down an electrochemical gradientof the GRC into the cell. An intracellular increase in the levels ofthis anion causes hyperpolarization of the transmembrane potential,rendering the neuron less susceptible to excitatory inputs (i.e.,reduced neuron excitability). In other words, the higher the chlorideion concentration in the neuron, the lower the brain excitability (thelevel of arousal).

It is well-documented that the GRC is responsible for the mediation ofanxiety, seizure activity, and sedation. Thus, GABA and drugs that actlike GABA or facilitate the effects of GABA (e.g., the therapeuticallyuseful barbiturates and benzodiazepines (BZs) such as Valium) producetheir therapeutically useful effects by interacting with specificregulatory sites on the GRC.

It has also been observed that a series of steroid metabolites interactwith the GRC to alter brain excitability (Majewska, M. D. et al.,“Steroid hormone metabolites are barbiturate-like modulators of the GABAreceptor,” Science 232:1004-1007 (1986); Harrison, N. L. et al.,Structure-activity relationships for steroid interaction with thegamma-aminobutyric acid-A receptor complex,” J. Pharmacol. Exp. Ther.241:346-353 (1987)). Prior to the present invention, the therapeuticusefulness of these steroid metabolites was not recognized by workers inthe field due to an incomplete understanding of the potency and site ofaction. Applicants' invention relates in part to a pharmaceuticalapplication of the knowledge gained from a more developed understandingof the potency and site of action of certain steroid compounds.

The ovarian hormone progesterone and its metabolites have beendemonstrated to have profound effects on brain excitability (Backstrom,T. et al., “Ovarian steroid hormones: effects on mood, behavior andbrain excitability,” Acta Obstet. Gynecol. Scand. Suppl. 130:19-24(1985); Pfaff, D. W. and McEwen, B. S., “Actions of estrogens andprogestins on nerve cells,” Science 219:808-814 (1983); Gyermek et al.,“Structure activity relationship of some steroidal hypnotic agents,” J.Med. Chem. 11:117 (1968); Lambert, J. et al., “Actions of synthetic andendogenous steroids on the GABA_(A) receptor,” Trends Pharmacol.8:224-227 (1987)). The levels of progesterone and its metabolites varywith the phases of the menstrual cycle. It has been well documented thatprogesterone and its metabolites decrease prior to the onset of menses.The monthly recurrence of certain physical symptoms prior to the onsetof menses has also been well documented. These symptoms, which havebecome associated with premenstrual syndrome (PMS) include stress,anxiety, and migraine headaches (Dalton, K., Premenstrual Syndrome andProgesterone Therapy, 2nd edition, Chicago: Chicago yearbook, 1984).Patients with PMS have a monthly recurrence of symptoms that are presentin premenses and absent in postmenses.

In a similar fashion, a reduction in progesterone has also beentemporally correlated with an increase in seizure frequency in femaleepileptics (i.e., catamenial epilepsy; Laidlaw, J., “Catamenialepilepsy,” Lancet, 1235-1237 (1956)). A more direct correlation has beenobserved with a reduction in progesterone metabolites (Rosciszewska etal., “Ovarian hormones, anticonvulsant drugs and seizures during themenstrual cycle in women with epilepsy,” J. Neurol. Neurosurg. Psych.49:47-51 (1986)). In addition, for patients with primary generalizedpetit mal epilepsy, the temporal incidence of seizures has beencorrelated with the incidence of the symptoms of premenstrual syndrome(Backstrom, T. et al., “Endocrinological aspects of cyclical moodchanges during the menstrual cycle or the premenstrual syndrome,” J.Psychosom. Obstet. Gynaecol. 2:8-20 (1983)). The steroiddeoxycorticosterone has been found to be effective in treating patientswith epileptic spells correlated with their menstrual cycles (Aird, R.B. and Gordan, G., “Anticonvulsive properties of deoxycorticosterone,”J. Amer. Med. Soc. 145:715-719 (1951)).

A syndrome also related to low progesterone levels is postnataldepression (PND). Immediately after birth, progesterone levels decreasedramatically leading to the onset of PND. The symptoms of PND range frommild depression to psychosis requiring hospitalization; PND isassociated with severe anxiety and irritability. PND-associateddepression is not amenable to treatment by classic antidepressants andwomen experiencing PND show an increased incidence of PMS (Dalton, K.,1984).

Collectively, these observations imply a crucial role for progesteroneand deoxycorticosterone and more specifically their metabolites in thehomeostatic regulation of brain excitability, which is manifested as anincrease in seizure activity or symptoms associated with catamenialepilepsy, PMS, and PND. The correlation between reduced levels ofprogesterone and the symptoms associated with PMS, PND, and catamenialepilepsy (Backstrom et al., 1983; Dalton, K., 1984) has prompted the useof progesterone in their treatment (Mattson et al., “Medroxyprogesteronetherapy of catamenial epilepsy,” in Advances in epileptology: XVthEpilepsy International Symposium, Raven Press, New York, 279-282, 1984,and Dalton, K., 1984). However, progesterone is not consistentlyeffective in the treatment of the aforementioned syndromes. For example,no dose-response relationship exists for progesterone in the treatmentof PMS (Maddocks, et al., “A double-blind placebo-controlled trial ofprogesterone vaginal suppositories in the treatment of premensturalsyndrome,” Obstet. Gynecol. 154:573-581 (1986); Dennerstein, et al.,British Medical Journal, 290:16-17 (1986)).

The publications and references referred to above and hereafter in thisspecification are incorporated herein by reference.

SUMMARY OF THE INVENTION

The present invention is directed to methods, compositions, andcompounds for modulating brain excitability. More particularly, theinvention relates to the use of 3α-hydroxylated steroid derivatives,acting at a newly identified site on the GR complex, to modulate brainexcitability in a manner that will alleviate stress, anxiety, insomnia,mood disorders (such as depression) that are amenable to GR-activeagents, and seizure activity. Compositions and compounds effective forsuch treatment are within the scope of the invention.

The compounds used in and forming part of the invention are modulatorsof the excitability of the central nervous system as mediated by theirability to regulate chloride ion channels associated with the GABA_(A)receptor complex. Applicants' experiments have established that thecompounds used in and of the invention have anticonvulsant andanxiolytic activity similar to the actions of known anxiolytic agentssuch as the BZs, but act at a distinct site on the GR complex.

The relationship of endogenous metabolites of progesterone to processesassociated with reproduction (estrus cycle and pregnancy) is wellestablished (Marker, R. E., Kamm, O., and McGrew, R. V., “Isolation ofepi-pregnanol-3-one-20 from human pregnancy urine,” J. Am. Chem. Soc.59:616-618 (1937)). Prior to the present invention, however, it was notrecognized how to treat disorders by modulating brain excitabilitythrough the use of progesterone metabolites and their derivatives.Therefore, this invention is directed to methods, compositions, andcompounds to treat disorders by modulating brain excitability using thecompounds of this invention. Representative disorders treated in thepresent invention are epilepsy, anxiety, pre-menstrual syndrome (PMS),post-natal depression (PND), mood disorders (such as depression) thatare amenable to GR-active agents, and insomnia. The compounds of theinvention can also be used to induce anesthesia.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention may be better understood and its advantagesappreciated by those skilled in the art by referring to the accompanyingdrawings wherein:

FIGS. 1A and 1B are plots of the percent binding of [³⁵S]t-butylbicyclophosphorothionate ([³⁵S] TBPS) to the cell membranes ofrat brain vs. log concentration of the alphaxalone (also calledalfaxalone) and GABA in various concentrations of (+)bicuculline.

FIG. 2 shows time courses for the dissociation of 2 nM [³⁵S] TBPS fromrat cortical P₂ homogenates initiated by the addition of 2 μM TBPS (▪),1 μM 3α5αP (□), 100 μM Na pentobarbital (), and 1 μM 3α5αP+100 μM Napentobarbital (∘).

FIG. 3 is a plot showing the effect of a single dosage of pentobarbitalon 3α-OH-5α-pregnan-20-one (3α-5α-P) modulation of [³H]-flunitrazepambinding in rat hippocampal homogenates.

FIG. 4 is a plot of the effect of 3α-hydroxy-5α-pregnan-20-one,3α,21-dihydroxy-5α-pregnan-20-one (5α-THDOC) and R5020 (promegesterone)on inhibiting [³⁵S] TBPS binding in rat cerebral cortex homogenate.

FIG. 5 is a plot of the correlation between TBPS binding andelectrophysiological activity of 15 different 3α-hydroxylated steroids.

FIG. 6 is a plot of the effect of 3α-OH-5α-pregnan-20-one,5α-pregnan-3α,20α-diol and 5β-pregnan-3α,20β-diol on inhibiting [³⁵S]TBPS binding in rat cortex homogenate.

FIG. 7 is a plot showing the effect of 3α-OH-5α-pregnan-20-one,5α-pregnan-3α,20α-diol, and 5β-pregnan-3α,20β-diol on the GABA-evokedcurrent in Xenopus oocytes injected with human recombinant GABA receptorsubunit α1β1γ2L.

FIG. 8 is a line graph of the number of transitions from light to darkoccurring within ten minutes of injection of 3α-OH-5α-pregnan-20-one and3α-OH-5β-pregnan-20-one.

FIGS. 9A and 9B are two line graphs of the percentage of (A) entriesinto and (B) the time on the open-arms during a five-minute test periodof 3α-OH-5α-pregnan-20-one and 3α-OH-5β-pregnan-20-one in the elevatedplus-maze test.

FIG. 10 is a line graph of the change in punished rats responding frombaseline for 3α-OH-5α-pregnan-20-one and 3α-OH-5β-pregnan-20-one in theGeller Seifter test.

FIG. 11 is a plot of the time course of anti-metrazol activity ofseveral prodrugs of 3α-OH-5α-pregnan-20-one.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The compounds of and used in the invention are derivatives of various3α-hydroxylated-pregnanes and 3α-hydroxylated-androstanes, and ester,ether, sulfonate, sulfate, phosphonate, phosphate, oxime, thiosulfate,heterocyclic and heteroaryl derivatives thereof, and derivativesreferred to as prodrugs. The expression “prodrug” denotes a derivativeof a known direct acting drug, which derivative has enhanced deliverycharacteristics and therapeutic value as compared to the drug, and istransformed into the active drug by an enzymatic or chemical process;see Notari, R. E., “Theory and Practice of Prodrug Kinetics,” Methods inEnzymology, 112:309-323 (1985); Bodor, N., “Novel Approaches in ProdrugDesign,” Drugs of the Future, 6(3):165-182 (1981); and Bundgaard, H.,“Design of Prodrugs: Bioreversible-Derivatives for Various FunctionalGroups and Chemical Entities,” in Design of Prodrugs (H. Bundgaard,ed.), Elsevier, New York (1985). It should be noted that some of thesynthetic derivatives forming part of the present invention may not betrue prodrugs because, in addition to the above characteristics, theyalso possess intrinsic activity. However, for purposes of thisapplication they will be referred to as prodrugs.

Our studies (Gee, K. W. et al., “GABA-dependent modulation of the Clionophore by steroids in rat brain,” European Journal of Pharmacology,136:419-423, 1987) have demonstrated that the 3α-hydroxylated steroidsused in the invention are orders of magnitude more potent than othershave reported (Majewska, M. D. et al. (1986) and Harrison, N. L. et al.(1987)) as modulators of the GR complex. Majewska et al. and Harrison etal. teach that the 3α-hydroxylated-5-reduced steroids are only capableof much lower levels of effectiveness. Our in vitro and in vivoexperimental data demonstrate that the high potency of these steroidsallows them to be therapeutically useful in the modulation of brainexcitability via the GR complex. The most potent steroids useful in thepresent invention include derivatives of major metabolites ofprogesterone and deoxycorticosterone. These steroids can be specificallyused to modulate brain excitability in stress, anxiety, insomnia, mooddisorders (such as depression) that are amenable to GR-active agents,and seizure disorders in a therapeutically beneficial manner.Furthermore, we have demonstrated that these steroids interact at aunique site on the GR complex which is distinct from other known sitesof interaction (i.e., barbiturate, BZ, and GABA) where therapeuticallybeneficial effects on stress, anxiety, sleep, mood disorders and seizuredisorders have been previously elicited (Gee, K. W. and Yamamura, H. I.,“Benzodiazepines and Barbiturates: Drugs for the Treatment of Anxiety,Insomnia and Seizure Disorders,” in In Central Nervous System Disorders,pages 123-147, D. C. Horvell, ed., 1985; Lloyd, K. G. and Morselli, P.L., “Psychopharmacology of GABAergic Drugs,” in Psychopharmacology: TheThird Generation of Progress, pages 183-195, H. Y. Meltzer, ed., RavenPress, N.Y., 1987). These compounds are desirable for their duration,potency and oral activity (along with other forms of administration).

The steroid derivatives of this invention are those having one of thefollowing structural formula (I):

wherein R, R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉ and R₁₀ are furtherdefined herein and the dotted lines are single or double bonds. Thestructure having Formula I includes androstanes, pregnanes (R₄=methyl),19-nor-androstanes, and norpregnanes (R₄=H).

The present invention also includes pharmaceutically acceptable estersand salts of the compounds of Formula I, including acid addition salts.It is believed that the 3α-hydroxyl may also be masked as apharmaceutically acceptable ester due to the fact that the ester will becleaved off as the prodrug is converted to drug form. These are referredto herein as cleavable esters.

Definitions

In accordance with the present invention and as used herein, thefollowing terms are defined with the following meaning, unlessexplicitly stated otherwise.

The term “alkyl” refers to saturated aliphatic groups including straightchain, branched chain, and cyclic groups, all of which may be optionallysubstituted. Suitable alkyl groups include methyl, ethyl, and the like,and may be optionally substituted.

The term “alkenyl” refers to unsaturated groups which contain at leastone carbon-carbon double bond and includes straight chain, branchedchain, and cyclic groups, all of which may be optionally substituted.

The term “alkynyl” refers to unsaturated hydrocarbon groups whichcontain at least one carbon-carbon triple bond and includes straightchain and branched chain groups which may be optionally substituted.Suitable alkynyl groups include propynyl, pentynyl, and the like whichmay be optionally substituted with cyano, acetoxy, halo, hydroxy orketo. Preferred alkynyl groups have five to eighteen carbon atoms. Morepreferred alkynyl groups have five to twelve carbon atoms. Mostpreferred alkynyl groups have five to seven carbon atoms.

The term “alkoxy” refers to the ether —OR wherein R is alkyl.

The term “aryloxy” refers to the ether —OR wherein R is aryl.

The term “aryl” refers to aromatic groups which have at least one ringhaving a conjugated pi electron system and includes carbocyclic aryl andbiaryl, both of which may be optionally substituted.

The term “carbocyclic aryl” refers to groups wherein the ring atoms onthe aromatic ring are carbon atoms. Carbocyclic aryl groups includephenyl and naphthyl groups optionally substituted. Substituted phenylhas preferably one to three, four or five substituents, such beingadvantageously, lower alkyl, amino, amido, cyano, carboxylate ester,hydroxy, lower alkoxy, halogen, lower acyl, and nitro.

The term “aralkyl” refers to an alkyl group substituted with an arylgroup. Suitable aralkyl groups include benzyl, and the like, and may beoptionally substituted.

The term “alkanoyloxy” refers to —O—C(O)R, wherein R is alkyl, alkenyl,alkynyl, aryl or aralkyl.

The term “carbalkoxyl” refers to —C(O)OR, wherein R is alkyl, alkenyl,alkynyl, aryl or aralkyl.

The term “carboxamido” refers to —C(O)NRR₁, wherein R and R₁ areindependently selected from hydrogen, alkyl, alkenyl, alkynyl, aryl oraralkyl.

The term “dialkylamino” refers to —NRR″ where R and R″ are independentlylower alkyl groups or together form the rest of a morpholino group.Suitable dialkylamino groups include dimethyl amino, diethylamino, andmorpholino.

The term “acyl” refers to the alkanoyl group —C(O)R where R is alkyl,alkenyl, alkynyl, aryl, or aralkyl.

The term “amino” refers to NRR′, wherein R and R′ are independentlyhydrogen, lower alkyl or are joined together to give a 5 or 6-memberedring, e.g. pyrrolidine or piperidine rings.

The term “optionally substituted” or “substituted” refers to groupssubstituted by one to three, four or five substituents, independentlyselected from lower alkyl (acylic and cyclic), aryl (carboaryl andheteroaryl), alkenyl, alkynyl, alkoxy, halo, haloalkyl (includingtrihaloalkyl, e.g. trifluoromethyl), amino, mercapto, alkylthio,alkylsulfinyl, alkylsulfonyl, nitro, alkanoyl, alkanoyloxy,alkanoyloxyalkanoyl, alkoxycarboxy, carbalkoxy (—COOR, wherein R islower alkyl), carboxamido (—CONRR′, wherein R and R′ are independentlylower alkyl), formyl, carboxyl, hydroxy, cyano, azido, keto and cyclicketals thereof, alkanoylamido, heteroaryloxy, heterocarbocyclicoxy, andhemisuccinate ester salts.

The term “lower” is referred to herein in connection with organicradicals or compounds defines such as one up to and including ten,preferably up to and including six, and advantageously one to fourcarbon atoms. Such groups may be straight chain, branched chain, orcyclic.

The term “heterocyclic” refers to carbon containing radicals havingfour, five, six, or seven membered rings and one, two or three O, N or Sheteroatoms, e.g., thiazolidine, tetrahydrofuran, 1,4-dioxane,pyrrolidine, piperidine, quinuclidine, dithiane, tetrahydropyran,ε-caprolactone, ε-caprolactam, ω-thiocaprolactam, and morpholine.

The term “heteroaryl” refers to carbon containing 5-14 membered cyclicunsaturated radicals containing one, two, three or four O, N or S atomsand having 6, 10 or 14 π electrons delocalized in one or more rings,e.g., pyridine, oxazole, indole, purine, pyrimidine, imidazole,benzimidazole, indazole, 2H-1,2,4-triazole, 1,2,3-triazole,2H-1,2,3,4-tetrazole, 1H-1,2,3,4-tetrazole, benzotriazole,1,2,3-triazolo[4,5-b]pyridine, thiazole, isoxazole, pyrazole, quinoline,cytosine, thymine, uracil, adenine, guanine, pyrazine, picolinic acid,picoline, furoic acid, furfural, furyl alcohol, carbazole,9H-pyrido[3,4-b]indole, isoquinoline, pyrrole, thiophene, furan,9(10H)-acridone, phenoxazine, and phenothiazine, each of which may beoptionally substituted as discussed above.

The term “dioic acids” refers to C₁₋₅ alkylene groups substituted withtwo carboxy groups, for example, malonic acid, succinic acid, glutaricacid, adipic acid, pimelic acid, and suberic acid. Hemi-ester salts ofthe dioic acids include the sodium, lithium, potassium, magnesium andcalcium salts thereof.

The term “β-acetyl-thiosulfate salt” refers is intended to include thesodium, lithium, potassium, magnesium and calcium salts thereof.

The term “pharmaceutically acceptable esters or salts” refers to estersor salts of Formula I derived from the combination of a compound of thisinvention and an organic or inorganic acid or base.

According to the present invention, ketals include diethers of loweralkanols, e.g. dimethyl and diethyl ketals, as well as cyclic ketalswhich include diethers of C₂₋₃ alkanediols, e.g. ethylene ketals andpropylene ketals.

Examples of substituents which can be used in the compounds of Formula Iare:

R is hydrogen, halogen, optionally substituted 1-alkynyl, lower alkoxy,alkyl, dialkylamino, or substituted alkyl;

R₁ is a substituted aralkynyl, arylalkyl, arylalkenyl, aryl, optionallysubstituted aralkylalkynyl, alkanoyloxyalkynyl, optionally substitutedheteroaryloxyalkynyl, oxoalkynyl or a ketal thereof, cyanoalkynyl,optionally substituted heteroarylalkynyl, hydroxyalkynyl, alkoxyalkynyl,aminoalkynyl, acylaminoalkynyl, mercaptoalkynyl, hydroxyalkynyl dioicacid hemi-ester or a salt thereof, or alkynyloxyalkynyl;

R₂ is hydrogen, hydroxy, alkoxy, alkanoyloxy, carbalkoxyl, a keto groupor amino group;

R₃ is an acetyl group, a ketal of an acetyl group; an alkoxyacetylgroup, an alkylthioacetyl group, an alkylsulfinylacetyl group, analkylsulfonylacetyl group, an aminoacetyl group, a trifluoroacetylgroup; a hydroxyacetyl group; an alkoxyalkylacetyl group, e.g. amethoxymethylacetyl group or an ethoxymethyl-2′-methylene acetyl group;a hydroxyalkyl group, e.g. a hydroxymethyl group, a 1′-hydroxyethylgroup, a 1′-hydroxypropyl group, or a 2′-hydroxy-2′-propyl group; ahydroxyacetyl dioic acid hemi-ester salt, e.g. a succinyloxyacetylgroup; an alkanoyloxyacetyl group, e.g. an acetoxyacetyl group; or asulfoxyacetyl group; an alkylacetyl group, e.g. a methylacetyl group; ahaloacetyl group; an ethynyl group; an optionally substitutedheteroarylacetyl group; an optionally substituted heteroaralkylacetylgroup which is also optionally substituted on the alkylene with ahydroxy, alkoxy, alkanoyloxy or carbalkoxyl group; an optionallysubstituted heterocyclic-acetyl group; an acetyl thiosulfate salt; acyano group; a alkylmethylene group (together with R₇); or analkoxymethylene group (together with R₇);

R₄ is hydrogen or methyl,

R₅ is hydrogen;

R₆ is hydrogen, alkanoyl, aminocarbonyl, or alkoxycarbonyl;

R₇ is hydrogen, halogen, hydroxy, alkoxy, alkanoyloxy, carbalkoxyl, amethylene group (together with R₃), or an alkoxymethylene group(together with R₃);

R₈ is hydrogen or halogen;

R₉ is hydrogen, halogen, alkyl, alkoxy, arylalkoxy or amino; and

R₁₀ is hydrogen, halogen, alkyl, haloalkyl, hydroxy, alkoxy,alkanoyloxy, carbalkoxyl, cyano, thiocyano or mercapto; provided thatwhen R₃ is an optionally substituted heteroarylacetyl group oracetylthiosulfate salts or when R is an optionally substituted 1-alkynylgroup, then R₁ may further be hydrogen, alkyl, alkenyl, aryl, aralkyl,alkynyl, optionally substituted aralkynyl, alkoxyalkyl, aminoalkyl,cyano, cyanoalkyl, thiocyanoalkyl, or azidoalkyl.

A preferred group of compounds of Formula I are compounds where R ishydrogen or lower alkoxy. More preferred are compounds where R, R₅, R₆,R₇, R₈, R₉ and R₁₀ are hydrogen.

Another group of preferred compounds are compounds of Formula I where R₁is substituted aralkynyl, e.g. R₁ is 4-substituted phenylalkynyl such as4-acetylphenylethynyl, 4-methoxyphenylethynyl,4-N,N-dimethylaminophenylethynyl, 4-cyanophenylethynyl,4-carboxyphenylethynyl ethyl ester, 4-N,N-dialkylamidophenylethynyl, orwhere R₁ is oxoalkynyl, hydroxyalkynyl, acetoxyalkynyl, cyanoalkynyl, oralkoxyalkynyl.

An additional group of preferred compounds are wherein R₃ is acetyl,heteroarylacetyl, heterocyclic-acetyl, hydroxyalkyl, hydroxyacetyl, andtheir esters with physiologically acceptable acids. More preferably, R₃is acetyl, β-succinyloxyacetyl, alkoxyacetyl, acetylthiosulfate salts,pyrazolylacetyl, or imidazolylacetyl.

An additional group of preferred compounds are wherein:

R is hydrogen, fluoro, chloro or lower alkoxy;

R₁ is substituted arylethynyl;

R₂ is hydrogen, a keto group or a dimethylamino group;

R₃ is a β-acetyl group, a dimethyl ketal of a β-acetyl group, atrifluoroacetyl group, a β-(hydroxyacetyl) group, aβ-methoxymethylacetyl group, a β-(ethoxy)methyl-2′-methylene acetylgroup, a β-(1′-hydroxyethyl) group, a β-(1′-hydroxypropyl) group, aβ-(2′-hydroxy-2′propyl) group, a β-succinyloxyacetyl group, aβ-hydroxyacetyl sodium succinate group, a β-acetoxyacetyl group, aβ-sulfoxyacetyl group, a β-methylacetyl group, a β-chloroacetyl group,or a β-ethynyl group;

R₄ is hydrogen or methyl;

R₅, R₆, R₇, R₈, R₉ and R₁₀ are hydrogen;

the dotted lines all represent bonds; and

R₇ is hydrogen or, when R₃ is β-hydroxyacetyl, R₇ is hydrogen orhydroxy.

Further preferred compounds are compounds of Formula I which are estersof hydroxyl groups at positions 3, 20 and/or 21. Preferred esters arethose obtained from their corresponding acids and dioic acids: acetic,propionic, maleic, fumaric, ascorbic, pimelic, succinic, glutaric,bismethylene-salicylic, methanesulfonic, ethane-di-sulfonic, oxalic,tartaric, salicylic, citric, gluconic, itaconic, glycolic,p-aminobenzoic, aspartic, glutamic, gamma-amino-butyric,α-(2-hydroxyethylamino)propionic, glycine and other α-amino acids,phosphoric, sulfuric, glucuronic, and 1-methyl-1,4-dihydronicotinic.

Preferred are the following compounds:3α-hydroxy-3β-phenylethynyl-5β-pregnan-20-one,3α-hydroxy-3β-phenylethynyl-5α-pregnan-20-one,3α-hydroxy-3β-(3′,4′-dimethoxyphenyl)ethynyl-5β-pregnan-20-one,3α-hydroxy-3β-(4′-methylphenyl)ethynyl-5β-pregnan-20-one,3α-hydroxy-3β-(2′-methoxyphenyl)ethynyl-5β-pregnan-20-one,3α-hydroxy-3β-(4′-carboxyphenyl)ethynyl-5β-pregnan-20-one ethyl ester,3α-hydroxy-3β-(4′-acetoxyacetylphenyl)ethynyl-5β-pregnan-20-one,3β-(4′-acetylphenyl)ethynyl-3α-hydroxy-5α-pregnan-20-one,3β-(4′-acetylphenyl)ethynyl-3α-hydroxy-5β-pregnan-20-one,3β-(4′-dimethylaminophenyl)ethynyl-3α-hydroxy-5β-pregnan-20-one,3β-(4′-biphenyl)ethynyl-3α-hydroxy-5β-pregnan-20-one,3α-hydroxy-3β-(4′-nitrophenyl)ethynyl-5β-pregnan-20-one,3α-hydroxy-3β-(4′-methoxyphenyl)ethynyl-5β-pregnan-20-one,3β-(4′-trifluoromethylphenyl)ethynyl-3α-hydroxy-5β-pregnan-20-one,3β-(4′-chlorophenyl)ethynyl-3α-hydroxy-5β-pregnan-20-one,3β-(4′-cyanophenyl)ethynyl-3α-hydroxy-5β-pregnan-20-one,3β-(4′(R/S)-hydroxypentynyl)-3α-hydroxy-5β-pregnan-20-one,3α-hydroxy-3β-phenyl-5β-pregnan-20-one,3α-hydroxy-3β-benzyl-5β-pregnan-20-one,3α-hydroxy-3β-(2′-phenylethyl)-5β-pregnan-20-one,3α-hydroxy-3β-[2-(3′,4′-dimethoxyphenyl)ethyl]-5β-pregnan-20-one,3α-hydroxy-3β-[6′-oxo-1′-heptynyl]-5β-pregnan-20-one,3α-hydroxy-3β-(7′-oxo-1′-octynyl)-5β-pregnan-20-one,3α-hydroxy-3β-(4′-oxo-1′-pentynyl)-5β-pregnan-20-one,3β-[5′-(R/S)-hydroxyhexynyl]-3α-hydroxy-5β-pregnan-20-one,3β-(4′-hydroxybutynyl)-3α-hydroxy-5β-pregnan-20-one,3β-(4′-hydroxybutynyl)-3α-hydroxy-5α-pregnan-20-one,3α-hydroxy-21-(1′-imidazolyl)-5β-pregnan-20-one,3α-hydroxy-3β-methyl-21-(1′,2′,4′-triazolyl)-5α-pregnan-20-one,3β-(4′-acetoxyphenylethynyl)-3α-hydroxy-5β-pregnan-20-one,3β-(4′-acetylphenylethynyl)-3α-hydroxy-19-nor-5β-pregnan-20-one,3β-(4′-carboxyphenylethynyl)-3α-hydroxy-19-nor-5β-pregnan-20-oneethylester,3β-(4′-carboxyphenylethynyl)-3α-hydroxy-5α-pregnan-20-oneethylester,3β-[4′-(N,N-diethylcarboxamido)phenyl]ethynyl-3α-hydroxy-5β-pregnan-20-one,3α-hydroxy-3β-[5-oxo-1-hexynyl]-5β-pregnan-20-one,3α-hydroxy-3β-[5′-oxo-1′-hexynyl]-5β-pregnan-20-one cyclic5′-(1,2-ethanediyl acetal),3β-(5-cyano-1-pentynyl)-3α-hydroxy-5β-pregnan-20-one,3α-hydroxy-3β-(2-pyridyl)ethynyl-5β-pregnan-20-one,3β-(6-hydroxy-1-hexynyl)-3α-hydroxy-5β-pregnan-20-one,3β-(6′-hydroxy-1′-hexynyl)-3α-hydroxy-5β-pregnan-20-one 6′-hemisuccinatesodium salt, 3β-(5′-hydroxy-1′-pentynyl)-3α-hydroxy-5β-pregnan-20-one,3β-(5′-hydroxy-1′-pentynyl)-3α-hydroxy-5β-pregnan-20-one5′-hemisuccinate sodium salt,3β-(4′-hydroxy-1′-butynyl)-3α-hydroxy-5β-pregnan-20-one 4′-hemisuccinatesodium salt, 3β-(4′-cyano-1′-butynyl)-3α-hydroxy-5β-pregnan-20-one,3β-(5′-acetoxy-1′-pentynyl)-3α-hydroxy-5β-pregnan-20-one,3β-(4′-acetoxy-1′-butynyl)-3α-hydroxy-5β-pregnan-20-one,3β-(4′-acetoxy-1′-butynyl)-3α-hydroxy-5α-pregnan-20-one,3β-(6′-acetoxy-1′-hexynyl)-3α-hydroxy-5β-pregnan-20-one,3α-hydroxy-3β-[3-(2′-propynyloxy)-1-propynyl]-5β-pregnan-20-one,3α-hydroxy-3β-(3-methoxy-1-propynyl)-5β-pregnan-20-one,3α-hydroxy-3β-(3-methoxy-1-propynyl)-5α-pregnan-20-one,3α-hydroxy-3β-[3-(4′-pyridinyloxy)-1-propynyl]-5β-pregnan-20-one,3α-hydroxy-3β-[3-(1′H-1,2,3-triazol-1′-yl)-1-propynyl]-5β-pregnan-20-one,3α-hydroxy-3β-[3-(2′H-1,2,3-triazol-2′-yl)-1-propynyl]-5β-pregnan-20-one,3α-hydroxy-3β-(2′-thienyl)ethynyl-5β-pregnan-20-one,3α-hydroxy-3β-(3′-phenyl-1′-propynyl)-5β-pregnan-20-one,3α-hydroxy-3β-(3′-phenylpropyl)-5β-pregnan-20-one,3α-hydroxy-3β-[3-(1′H-pyrazol-1′-yl)-1-propynyl]-5β-pregnan-20-one,3β-(3′-acetylphenylethynyl)-3α-hydroxy-5β-pregnan-20- one,3β-(3′-acetoxy-3′-propynyl)-3α-hydroxy-5β- pregnan-20-one,3α-hydroxy-3β-(4-hydroxybutyn-1-yl)-21-(1-imidazolyl)-5β-pregnan-20-one,3α-hydroxy-3β-(4-hydroxybutyn-1-yl)-21-(1-imidazolyl)-5β-19-nor-pregnan-20-one,3α-hydroxy-3β-(4-hydroxybutyn-1-yl)-21-(1,2,3-triazol-2-yl)-5β-19-norpregnan-20-one,3α-hydroxy-21-(1-imidazolyl)-5α-pregnan-20-one, sodiumS-(3α-hydroxy-3β-methyl-5α-pregnan-20-on-21-yl) thiosulfate, sodiumS-(3α-hydroxy-3β-methoxymethyl-5α-pregnan-20-on-21-yl) thiosulfate,sodium S-3α-hydroxy-5β-pregnan-20-on-21-yl) thiosulfate, sodiumS-(3α-hydroxy-3β-trifluoromethyl-5β-pregnan-20-on-21-yl) thiosulfate,sodiumS-[3α-hydroxy-3β-(4′-hydroxybutynyl)-5β-pregnan-20-on-21-yl]thiosulfate,sodium-S-(3α-hydroxy-5α-pregnan-20-on-21-yl) thiosulfate, and3β-(4′-acetylphenylethynyl)-3α,21-dihydroxy-5β-pregnan-20-one,3β-(4′-acetylphenylethynyl)-3α,21-dihydroxy-5α-pregnan-20-one,3β-(4′-acetylphenylethynyl)-3α,21-dihydroxy-5β-pregnan-20-one21-hemisuccinate sodium salt, and3β-(4′-acetylphenylethynyl)-3α,21-dihydroxy-5α-pregnan-20-one21-hemisuccinate sodium salt.

The more preferred neuroactive steroids include 3β-(4′-acetylphenyl)ethynyl-3α-hydroxy-5α-pregnan-20-one,3β-(4′-carboxylphenyl)ethynyl-3α-hydroxy-5α-pregnan-20-one ethyl ester,3β-(4′-acetylphenyl) ethynyl-3α-hydroxy-5β-pregnan-20-one,3β-(4′-carboxylphenyl) ethynyl-3′-hydroxy-5β-pregnan-20-one ethyl ester,3β-(4′-acetylphenyl) ethynyl-3α-hydroxy-5β-19-norpregnan-20-one,3β-(4′-carboxylphenyl) ethynyl-3α-hydroxy-5β-19-norpregnan-20-one ethylester, 3β-(4′-dimethylaminophenyl) ethynyl-3α-hydroxy-5β-pregnan-20-one,3β-(4′-biphenyl) ethynyl-3α-hyroxy-5β-pregnan-20-one,3α-hydroxy-3β-(4′-methoxyphenyl)ethynyl-5β-pregnan-20-one,3β-(4′-trifluoromethylphenyl) ethynyl-3α-hydroxy-5β-pregnan-20-one,3β-(4′-chlorophenyl) ethynyl-3α-hydroxy-5β-pregnan-20-one,3β-[4′(R/S)-hydroxypentynyl]-3α-hydroxy-5β-pregnan-20-one,3β-(4′-hydroxybutynyl)-3α-hydroxy-5β-pregnan-20-one;3β-(4′-hydroxybutynyl)-3α-hydroxy-5α-pregnan-20-one;3α-hydroxy-3β-[3-(2′H-1,2,3-triazol-2′-yl)-1-propynyl]-5β-pregnan-20-one;3α-hydroxy-21-(1-imidazolyl)-5β-pregnan-20-one,3β-(4′-acetylphenylethynyl)-3α,21-dihydroxy-5β-pregnan-20-one,3β-(4′-acetylphenylethynyl)-3α21-dihydroxy-5α-pregnan-20-one,3β-(4′-acetylphenylethynyl)-3α21-dihydroxy-5β-pregnan-20-one21-hemisuccinate sodium salt, and3β-(4′-acetylphenylethynyl)-3α,21-dihydroxy-5α-pregnan-20-one21-hemisuccinate sodium salt.

The especially preferred neuoroactive steroids include3β-(4′-acetylphenyl) ethynyl-3α-hydroxy-5α-pregnan-20-one,3β-(4′-acetylphenyl) ethynyl-3α-hydroxy-5β-pregnan-20-one,3β-(4′-carboxylphenyl) ethynyl-3α-hydroxy-5α-pregnan-20-one ethyl ester,3β-(4′-carboxylphenyl) ethynyl-3α-hydroxy-5β-pregnan-20-one ethyl ester,3β-(4′-dimethylaminophenyl)ethynyl-5β-pregnan-20-one,3β-(4′-biphenyl)ethynyl-3α-hydroxy-5β-pregnan-20-one,3β-(4′-hydroxybutynyl)-3α-hydroxy-5β-pregnan-20-one3β-(4′-hydroxybutynyl)-3α-hydroxy-5α-pregnan-20-one;3α-hydroxy-3β-[3-(2′H-1,2,3-triazol-2′-yl)-1-propynyl]-5β-pregnan-20-one;3α-hydroxy-21-(1-imidazolyl)-5β-pregnan-20-one,3β-(4′-acetylphenylethynyl)-3α,21-dihydroxy-5β-pregnan-20-one,3β-(4′-Acetylphenylethynyl)-3α21-dihydroxy-5α-pregnan-20-one,3β-(4′-Acetylphenylethynyl)-3α,21-dihydroxy-5β-pregnan-20-one21-hemisuccinate sodium salt,3β-(4′-Acetylphenylethynyl)-3α,21-dihydroxy-5α-pregnan-20-one21-hemisuccinate sodium salt and3β-[4′(R/S)-hydroxypentynyl]-3α-hydroxy-5β-pregnan-20-one.

SYNTHETIC METHODS

The compounds according to the invention may be prepared by anyconvenient method, e.g. using conventional techniques such as aredescribed in “Steroid Reactions,” Djerassi, published in 1963 byHolden-Day, Inc., San Francisco or “Organic Reactions in SteroidChemistry”, Fried and Edwards, published in 1972 by VanNostrand-Reinhold Co., New York.

GENERAL METHODS

20-hydroxy pregnanes were prepared by the reduction of 20-keto pregnaneswith conventional reducing agents.

21-Hemisuccinates were prepared from pregnan-20-one derivatives whichwere first brominated with molecular bromine to obtain the corresponding21-bromo pregnanes. The bromo compounds were then reacted with variousdioic acids, such as succinic acid, in the presence of an amine to yield21-hydroxy esters. The resulting esters from the dioic acids were thenconverted to their sodium salts by conventional means.

21-Oxygenated compounds of this type may be prepared by a reactionsequence in which a pregnan-20-one is oxidized with lead tetraacetate togive a 21-acetoxy derivative, hydrolysis of the acetate to give a21-alcohol, and acylation with an appropriate carboxylic acidderivative, for example, an anhydride or acid chloride or other reagentcapable of replacing the hydrogen of the hydroxyl group, such asmethanesulfonyl chloride.

Pregn-17-enes may be formed by the reaction of a 17-ketosteroid with aWittig reagent such as the ylide derived from treatment ofn-propyltriphenylphosphonium bromide with a strong base such aspotassium t-butoxide.

EXAMPLE 1 3α-Hydroxy-3β-(2′-phenylethyl)-5β-pregnan-20-one

A solution of 3α-hydroxy-3β-phenylethynyl-5β-pregnan-20-one (44 mg) wasdissolved in EtOAc (12 mL), Pd/C (5%, 12 mg) was added and the mixturewas hydrogenated at 400 Kpa pressure overnight at rt. filtration of thecatalyst followed by evaporation of the solvent yielded the crudeproduct, which was purified by chromatography over silica gel to isolatethe pure title compound (33 mg); mp 153-154° C.; TLC R_(f)(hexane:acetone 7:3)=0.4.

EXAMPLE 2 3α-(3′,4′-Dimethoxyphenylethynyl)-3β-hydroxy-5β-pregnan-20-oneand 3β-(3′,4′-Dimethoxyphenylethynyl)-3α-hydroxy-5β-pregnan-20-one

A solution of 2,2-dibromo-1-(3′,4′-dimethoxyphenyl)ethene (prepared bythe Wittig reaction of 3,4-dimethoxybenzaldehyde with carbontetrabromide in the presence of triphenyl phosphine) (966 mg, 3 mmol) indry THF (15 mL) was treated under N₂ with n-BuLi (2.5M in THF, 6 mmol,2.4 mL) at −78° C. The mixture was stirred at this temperature for 2hours and a solution of 5β-pregnan-3,20-dione cyclic 20-(1,2-ethanediylacetal) (720 mg, 2 mmol) in dry THF (10 mL) was added dropwise over aperiod of 30 min. After stirring the resulting mixture at −78° C. for 2hr, the cooling bath was removed and the stirring was continued at rtfor another hr. It was then quenched with 2N HCl solution (1 mL) at −10°C. The solvent was removed and the residue was then dissolved in acetone(25 mL). After adding 2N HCl (10 mL) the solution was stirred at rt for2 hr. Sat. NaHCO₃ soln. was added to neutralize the acid. The solventswere removed and the residue was extracted with EtOAc. The organic layerwas washed with water, dil. NaHCO₃ soln., water, and brine. After dryingover anhyd. MgSO₄ the solution was filtered and evaporated to yield thecrude product (1.2 g). This crude product was then dissolved in a smallamount of CH₂Cl₂ and poured on a column of silica gel. Elution with thetoluene:acetone mixture (96:4) gave a phenylacetylene compound, whichwas not characterized. Further elution with the same solvent yielded3α-(3′,4′-dimethoxyphenylethynyl)-3β-hydroxy-5β-pregnan-20-one (120 mg)as a first fraction, and3β-(3′,4′-dimethoxyphenylethynyl)-3α-hydroxy-5β-pregnan-20-one as asecond fraction (430 mg); mp 82-88° C.

An analogous method was used to prepare:3β-(4′-methoxyphenylethynyl)-3α-hydroxy-5β-pregnan-20-one;3β-(4′-chlorophenylethynyl)-3α-hydroxy-5β-pregnan-20-one;3β-(2′-methoxyphenylethynyl)-3α-hydroxy-5β-pregnan-20-one;3β-(4′-biphenylethynyl)-3α-hydroxy-5β-pregnan-20-one;3β-(4′-dimethylaminophenylethynyl)-3α-hydroxy-5β-pregnan-20-one; and3β-(4′-cyanophenylethynyl)-3α-hydroxy-5β-pregnan-20-one.

EXAMPLE 3 3β-(3′,4′-dimethoxyphenylethyl)-3α-hydroxy-5β-pregnan-20-one

A mixture of Pd/C (5%, 28 mg) and EtOAc (12 mL) was presaturated withhydrogen by stirring it under hydrogen for 10 min. A solution of3β-(3′,4′-dimethoxyphenylethynyl)-3α-hydroxy-5β-pregnan-20-one (185 mg)in EtOAc (5 mL) was then added and the mixture was hydrogenated at 300Kpa pressure overnight at rt. Filtration of the catalyst followed byevaporation of the solvent yielded the crude product, which was purifiedby chromatography over silica gel (hexane:acetone 4:1) to isolate thepure title compound (135 mg); TLC R_(f) (hexaneacetone 4:1)=0.14.

EXAMPLE 4 3β-(4′-Nitrophenylethynyl)-3α-hydroxy-5β-pregnan-20-one

A solution of 2,2-dibromo-1-(4-nitrophenyl)ethene (prepared by theWittig reaction of 4-nitrobenzaldehyde with carbon tetrabromide in thepresence of triphenyl phosphine) (296 mg, 1 mmol) in dry THF (20 mL) wastreated under N₂ with n-BuLi (2.5M in THF, 2 mmol, 0.8 mL) at −95° C.The mixture was stirred at −80 to −100° C. for 0.5 hr and then asolution of 5β-pregnan-3,20-dione cyclic 20-(1,2-ethanediyl acetal) (120mg, 0.5 mmol) in dry THF (10 mL) was added dropwise over a period of 10min. After stirring the resulting mixture at −80° C. for 1 hr, and thenat 0° C. for 1 more hr it was quenched with NH₄Cl solution (3 mL). Thesolvent was removed and the residue was then dissolved in acetone (25mL). After adding 2N HCl (10 mL) the solution was stirred at rt for 1hr. Saturated NaHCO₃ soln. was added to neutralize the acid. Thesolvents were removed and the residue was extracted with CH₂Cl₂. Theorganic layer was washed with water, and brine. After drying over anhyd.MgSO₄ the solution was filtered and evaporated to yield the crudeproduct (400 mg). This crude product was then dissolved in a smallamount of CH₂Cl₂ and poured on a column of silica gel. Elution withtoluene:acetone mixture (96:4) gave the title compound as a brown solid(70 mg); TLC R_(f) (toluene:acetone 95:5)=0.18.

EXAMPLE 5 3β-Hydroxy-3α-phenyl-5β-pregnan-20-one and3α-hydroxy-3β-phenyl-5β-pregnan-20-one

A solution of 5β-pregnan-3,20-dione cyclic 20-(1,2-ethanediyl acetal)(720 mg, 2 mmol) in 15 ml of dry THF was treated with phenyl magnesiumbromide (3M in THF, 6 mmol, 2 mL) at −70° C. After stirring the mixtureat this temperature for 3 hr and then at rt for 2 hr, it was quenchedwith 2N HCl (1mL). The solvent was removed and the residue was dissolvedin acetone (20 mL). After adding 1N HCl (5 mL) the solution was stirredat rt for 15 hr. The solvents were removed and the residue was extractedwith CH₂Cl₂. The organic layer was washed with water, dil. NaHCO₃ soln.,water, and brine. After drying over anhyd. MgSO₄ the solution wasfiltered and evaporated to yield the crude product (1.3 g). This crudeproduct was then dissolved in a small amount of CH₂Cl₂ and poured on acolumn of silica gel. Elution with toluene:acetone mixture (95:5) gave a3α-phenyl-3β-hydroxy-5β-pregnan-20-one (420 mg) as a first fraction.Further elution with the same solvent mixture yielded3β-phenyl-3α-hydroxy-5β-pregnan-20-one (185 mg), m.p. 182-184° C.

EXAMPLE 6 3β-Hydroxy-3α-benzyl-5β-pregnan-20-one and3α-hydroxy-3β-benzyl-5β-pregnan-20-one

A solution of benzyl magnesium bromide (2M in THF, 2 mmol, 1 mL) wasdiluted with THF (15 mL) and was treated dropwise with a solution of5β-pregnan-3,20-dione cyclic 20-(1,2-ethanediyl acetal) (360 mg, 1 mmol)in dry THF (15 mL) at −60° C. After stirring the mixture at thistemperature for 1 hr and then at rt for 15 hr, it was quenched with 2NHCl (1 mL). The solvent was removed and the residue was dissolved inacetone (20 mL). After adding 1N HCl (5 mL) the solution was stirred atrt for 30 min. It was neutralized with 2N NaOH. The precipitated solidwas collected by filtration, washed with water and dried to yield3β-hydroxy-3α-benzyl-5β-pregnan-20-one (238 mg). The filtrate wasextracted with EtOAc. The organic layer was washed with water, dil.NaHCO₃ soln., water, and brine. After drying over anhyd. MgSO₄ thesolution was filtered and evaporated to give the crude product (160 mg).This crude product was then dissolved in a small amount of CH₂Cl₂ andpoured on a column of silica gel. Elution with toluene:acetone mixture(95:5) gave 3β-hydroxy-3α-benzyl-5β-pregnan-20-one (40 mg) as a firstfraction. Further elution with the same solvent mixture yielded3α-hydroxy-3β-benzyl-5β-pregnan-20-one (30 mg), which was crystallizedfrom hexane:CH₂Cl₂ (4:1) as colorless rods (15 mg); m.p. 133-141° C.;TLC R_(f) (toluene:acetone 9:1)=0.5.

EXAMPLE 7 3β-[3′(R/S)-Hydroxybutynyl]-3α-hydroxy-5β-pregnan-20-one

A solution of 3 (R/S)-hydroxybutyne (0.470 mL, 6 mmol) in dry THF (15mL) was treated with n-BuLi (2.5M in THF, 12 mmol, 4.8 mL) at −70° C.After stirring the mixture at this temperature for 0.5 hr. a solution of5β-pregnan-3,20-dione cyclic 20-(1,2-ethanediyl acetal) (1.08 g, 3 mmol)in dry THF (30 mL) was added and the mixture was stirred at −78° C. for1 hr. The cooling bath was removed and the mixture was stirred at rt foranother 1.5 hr. It was then quenched with NH₄Cl solution (3 mL). Thesolvent was removed and the residue was the dissolved in acetone (10mL). After adding 2N HCl (5 mL), the solution was stirred at rt for 1hr. Saturated NaHCO₃ soln. was added to neutralize the acid. Thesolvents were removed and the residue was extracted with EtOAc. Theorganic layer was washed with water, dil. NaHCO₃ soln., water, andbrine. After drying over anhyd. MgSO₄ the solution was filtered andevaporated to yield the crude product (1.4 g). This crude product wasthen dissolved in a small amount of CH₂Cl₂ and poured on a column ofsilica gel. Elution with toluene:acetone mixture (85:15) gave3β-[3′(RS)-hydroxybutynyl]-3α-hydroxy-5β-pregnan-20-one (145 mg) as acolorless solid; TLC R_(f) (toluene:acetone 4:1)=0.24.

An analogous method was used to prepare:3β-[4′(R/S)-hydroxypentynyl]-3α-hydroxy-5β-pregnan-20-one.

EXAMPLE 8 3β-(4′-Acetylphenylethynyl)-3α-hydroxy-5α-pregnan-20-one

A solution of 4-iodoacetophenone (95 mg, 0.39 mmol),3β-ethynyl-3α-hydroxy-5α-pregnan-20-one (106 mg, 0.3 mmol) in drydegassed pyrrolidine (3 mL) was stirred under argon at rt.Bis(triphenylphosphine)palladium (II) dichloride (5 mg) and CuI (5 mg)were added and the mixture was stirred at rt for 15 hr. The TLC showed100% conversion of the starting material, hence, the mixture wasquenched with NH₄Cl solution (15 mL) and was extracted with EtOAc. Theorganic layer was washed with water, dil. NaHCO₃ soln., water, andbrine. After drying over anhyd. MgSO₄ the solution was filtered andevaporated to yield the crude product (150 mg). This crude product wasthen dissolved in a small amount of CH₂Cl₂ and poured on a column ofsilica gel. Elution with hexane:acetone mixture (4:1) afforded3β-(4′-acetylphenylethynyl)-3α-hydroxy-5α-pregnan-20-one(35 mg) as acolorless solid; TLC R_(f) (hexane:acetone 7:3)=0.4.

An analogous method was used to prepare:3α-hydroxy-3β-(4′-trifluoromethylphenylethynyl)-5β-pregnan-20-one;3β-(4′-acetylphenylethynyl)-3α-hydroxy-5β-pregnan-20-one;3α-hydroxy-3β-(4′-methylphenylethynyl)-5β-pregnan-20-one;3α-hydroxy-3β-(4α-acetoxyacetylphenylethynyl)-5β-pregnan-20-one;3β-(3′-hydroxyphenylethynyl)-3α-hydroxy-5β-pregnan-20-one;3β-(2′,4′-difluorophenylethynyl)-3α-hydroxy-5β-pregnan-20-one;3β-(pentafluorophenylethynyl)-3α-hydroxy-5β-pregnan-20-one; and3α-hydroxy-3β-(4′-carboxyphenylethynyl)-5β-pregnan-20-one ethyl ester.

EXAMPLE 9 3α-Hydroxy-21-(1′-imidazolyl)-5β-pregnan-20-one

3α-Hydroxy-21-bromo-5β-pregnan-20-one: To a flask containing a solutionof 3α-hydroxy-5β-pregnan-20-one (5.15 g, 16.5 mmol) in methanol (100 mL)was added a solution of bromine (1.1 mL) in methanol (30 mL) dropwise insuch a rate to maintain the brown color of the bromine until this colorwas persistent. Then water (200 mL) was added and the mixture wasextracted with CH₂Cl₂ (3×100 mL). The combined extracts were dried overNa₂SO₄. Removal of the solvent resulted in the product as a foamy whitesolid (6.63 g). Other 21-bromo-pregnan-20-ones(3β-ethynyl-3α-hydroxy-21-bromo-5β-pregnan-20-one,3β-methyl-3α-hydroxy-21-bromo-5α-pregnan-20-one and3α-hydroxy-3β-trifluoromethyl-12-bromo-5β-19-norpregnan-20-one) weresynthesized using the same method.

3α-Hydroxy-21-(1′-imidazolyl)-5β-pregnan-20-one: A mixture of3α-hydroxy-21-bromo-5β-pregnan-20-one (0.86 g) and imidazole (0.378 g)in CH₃CN (12 mL) was heated under Ar to reflux for 1 h and cooled to 25°C. It was then poured into a separatory funnel containing NH₄Cl solution(100 mL, aq. sat.) and the product was extracted with EtOAc (3×50 mL).The combined organics were dried over Na₂SO₄ and the solvent was removedin vacuo. The pure product (0.59 g, 42%) was isolated by flash columnchromatography.

The compounds 3α-hydroxy-21-(1′-benzimidazolyl)-5β-pregnan-20-one,3α-hydroxy-21-[1H-(4-methyl-5-carbethoxyl)imidazol-1-yl)-5β-pregnan-20-oneethylester, 3α-hydroxy-21-(1′-imidazolyl)-5α-pregnan-20-one,3β-ethynyl-3α-hydroxy-21-(1′-imidazolyl)-5β-pregnan-20-one,3α-hydroxy-21-(1H-3,5-dimethylpyrazolyl)-5β-pregnan-20-one,3α-hydroxy-21-(1′-imidazolyl)-3β-methyl-5α-pregnan-20-one,3α-hydroxy-21-(1′-pyrazolyl)-5α-pregnan-20-one,3β-ethynyl-3α-hydroxy-21-(1′-pyrazolyl)-5β-pregnan-20-one,21-(1′-benzimidazolyl)-3α-hydroxy-3β-methyl-5α-pregnan-20-one,3α-hydroxy-21-(1′-pyrazolyl)-3β-methyl-5α-pregnan-20-one,3α-hydroxy-21-(pyrazol-1-yl)-5β-pregnan-20-one,3α-hydroxy-21-[1H-(2-methyl)imidazol-1-yl)]-5β-pregnan-20-one,3α-hydroxy-21-[1H-(2′-formyl)imidazol-1-yl)]-5β-pregnan-20-one,3α-hydroxy-21-(1H-imidazol-1-yl)-3β-trifluoromethyl-5β-19-norpregnan-20-one,and3α-hydroxy-21-(pyrazol-1-yl)-3β-trifluoromethyl-5β-19-norpregnan-20-onewere synthesized according to Example 9.

EXAMPLE 103α-Hydroxy-3β-methyl-21-(1′,2′,4′-triazolyl)-5α-pregnan-20-one:

To a solution of 1,2,4-triazole (146 mg) in THF (15 mL) under Ar wasadded NaH (56 mg) and the mixture obtained was stirred at 25° C. for 20min. Then a solution 3β-methyl-3α-hydroxy-21-bromo-5α-pregnan-20-one(300 mg) in THF was added and the mixture thus obtained was stirred for5 hr. It was then poured into a separatory funnel containing water (40mL) and the product was extracted with CH₂Cl₂ (3×50 mL). The combinedorganics were dried over Na₂SO₄ and the solvent was removed in vacuo.The pure product (187 mg, 64%) was isolated by flash columnchromatography.

3α-Hydroxy-21-(2′H-1,2,3,4-tetrazol-2′-yl)-5β-pregnan-20-one,3α-hydroxy-21-(2H-1,2,3-triazol-2-yl)-5β-pregnan-20-one,3α-hydroxy-21-(9′H-purin-9′-yl)-5β-pregnan-20-one,3α-hydroxy-3β-methyl-21-(2′-H-1′,2′,3′-triazol-2′-yl)-5α-pregnan-20-one,3β-ethynyl-3α-hydroxy-21-(1′,2′,4′-triazolyl)-5β-pregnan-20-one,3α-hydroxy-3β-methyl-21-(1′,2′,3′-triazol-1′-yl)-5α-pregnan-20-one,3α-hydroxy-21-(1′,2′,4′-triazol-1-yl)-5β-pregnan-20-one,3α-hydroxy-21-(1′H-1,2,3,4-tetrazol-1′-yl)-5β-pregnan-20-one,3α-hydroxy-21-[1H-(4-nitro) imidazol-1-yl)-5β-pregnan-20-one,3α-hydroxy-21-(7′H-purin-7′-yl)-5β-pregnan-20-one,3α-hydroxy-21-[1H-(4′,5′-dicyano) imidazol-1-yl)]-5β-pregnan-20-one,3α-hydroxy-21-(1′-1,2,4-triazol-1-yl)-3β-trifluoromethyl-5β-19-norpregnan-20-one,and 3α-hydroxy-21-[1H-(4′,5′-dichloro)imidazol-1-yl)]-5β-pregnan-20-one,were synthesized according to the procedures set forth in Example 10.

EXAMPLE 11 3β-(4′-Acetoxyphenylethynyl)-3α-hydroxy-5β-pregnan-20-one a.3β-(4′-Hydroxyphenylethynyl)-3α-hydroxy-5β-pregnan-20-one

A solution of 2,2-dibromo-1-(4-hydroxyphenyl)ethene (prepared by theWittig reaction of 4-hydroxybenzaldehyde with carbon tetrabromide in thepresence of triphenyl phosphine) (1.25 mg, 4.5 mmol) in dry THF (25 mL)was treated under N₂ with n-BuLi (2.5M in THF, 13.5 mmol, 5.4 mL) at 70°C. The mixture was then stirred at −70° C. temp. for 0.5 hr, and asolution of 5β-pregnan-3,20-dione, cyclic 20-(1,2-ethanediyl acetal)(810 mg, 2.25 mmol) in dry THF (25 mL) was added dropwise over a periodof 10 min. After stirring the resulting mixture at −78° C. for 30 min,the cooling bath was removed and the stirring was continued at rt foranother hr. It was then quenched with sat. NH₄Cl solution (4 mL) at −10°C. The solvent was removed and the residue was then dissolved in acetone(25 mL). After adding 2N HCl (6 mL) the solution was stirred at rt for15 min. Sat. NaHCO₃ soln. was added to neutralize the acid. The solventwas removed under reduced pressure and the crude product (1.3 g) wasused as such for the next step.

b. 3β-(4′-Acetoxyphenylethynyl)-3α-hydroxy-5β-pregnan-20-one

The crude product from the above step was dissolved in CH₂Cl₂ (5 mL) andpyridine (3 mL). The resulting solution was added to a ice-cold mixtureof acetyl chloride (3 mL) and pyridine (3 mL). The mixture was stirredat 0° C. for 30 min. and was poured into ice (50 g). 2N HCl (10 mL) wasadded and the mixture was diluted with more CH₂Cl₂.The organic layer wasseparated, washed with water, brine, and dried over anhyd. MgSO₄.Removal of solvent gave a crude solid (2 g) which was purified by columnchromatography over silica gel. Elution with tolueneacetone mixture(95:5) gave 3α-(4′-acetoxyphenylethynyl)-3β-hydroxy-5β-pregnan-20-one(150 mg). Further elution with the same solvent mixture yielded3β-(4′-acetoxyphenylethynyl)-3α-hydroxy-5β-pregnan-20-one (530 mg); m.p.171-176° C.; TLC R_(f) (hexaneacetone 7:3)=0.41.

EXAMPLE 123β-(4′-Acetylphenylethynyl)-3α-hydroxy-19-nor-5β-pregnan-20-one

A solution of 4-iodoacetophenone (60 mg, 0.24 mmol),3β-ethynyl-3α-hydroxy-19-nor-5β-pregnan-20-one (80 mg, 0.24 mmol) in drydegassed triethylamine (0.5 mL) was stirred under argon at 23° C.Bis(triphenylphosphine)-palladium(II)dichloride (5 mg) and CuI (5 mg)were added and the mixture was stirred at this temp. for 45 min. CH₂Cl₂(4 mL) was added and the mixture was stirred at 23° C. for 3 hr. The TLCshowed 100% conversion of the starting material, hence, the solvent wasremoved and the residue was purified by chromatography on silica gel.Elution with hexane:acetone (4:1) gave3β-(4′-acetylphenyl-ethynyl)-3α-hydroxy-19-nor-5β-pregnan-20-one (30 mg)as a colorless solid; mp 65-67° C., TLC R_(f) (hexaneacetone 4:1)=0.12.

EXAMPLE 133β-(4′-Carboxyphenylethynyl)-3α-hydroxy-19-nor-5β-pregnan-20-one ethylester

A solution of ethyl 4-iodobenzoate (70 mg, 0.24 mmol) and3β-ethynyl-3α-hydroxy-19-nor-5β-pregnan-020-one (80 mg, 0.24 mmol) indry degassed triethylamine (1 mL) was stirred under argon at 23° C.Bis(triphenylphosphine)-palladium(II)dichloride (5 mg) and CuI (5 mg)were added and the mixture was stirred at this temp. for 1 hr. CH₂Cl₂ (4mL) was added and the mixture was stirred at 23° C. for 3 hr. The TLCshowed 100% conversion of the starting material, hence, the solvent wasremoved and the residue was purified by chromatography on silica gel.Elution with hexane:acetone (4:1) gave3β-(4′-carboxyphenylethynyl)-3α-hydroxy-19-nor-5β-pregnan-20-one ethylester (22 mg) as a colorless solid; mp 164-166° C., TLC R_(f)(hexane:acetone 4:1)=0.27.

EXAMPLE 14 3β-(4′-Carboxyphenylethynyl)-3α-hydroxy-5α-pregnan-20-oneethyl ester

A solution of ethyl 4-iodobenzoate (83 mg, 0.3 mmol),3β-ethynyl-3α-hydroxy-5α-pregnan-20-one (103 mg, 0.3 mmol) in drydegassed triethylamine (1 mL) was stirred under argon at 23° C.Bis(triphenylphosphine)-palladium (II)dichloride (5 mg) and CuI (5mg)were added and the mixture was stirred at this temp. for 1 hr. CH₂Cl₂ (4mL) was added and the mixture was stirred at 23° C. for 1.5 hr. The TLCshowed 100% conversion of the starting material, hence, the solvent wasremoved and the residue was purified by chromatography on silica gel.Elution with hexane:acetone (4:1) gave3β-(4′-carboxyphenylethynyl)-3α-hydroxy-5α-pregnan-20-one ethylester (10mg) as a colorless solid; mp 190-192° C., TLC R_(f) (hexane:acetone4:1)=0.27.

EXAMPLE 153β-[4′-(N,N-diethylcarboxamido)phenyl]ethynyl-3α-hydroxy-5β-pregnan-20-one

A solution of 4-(N,N-diethylcarboxamido)iodobenzene (91 mg, 0.3 mmol),3β-ethynyl-3α-hydroxy-5β-pregnan-20-one (103 mg, 0.3 mmol) in drydegassed triethylamine (1 mL) was stirred under argon at 23° C.Bis(triphenylphosphine) palladium(II)dichloride (5 mg) and CuI (5 mg)were added and the mixture was stirred at this temp. for 1 hr. CH₂Cl₂ (4mL) was added and the mixture was stirred at 23° C. for 0.5 hr. The TLCshowed 100% conversion of the starting material, hence, the solvent wasremoved and the residue was purified by chromatography on silica gel.Elution with hexane:acetone (3:1) gave3β-[4′-(N,N-diethylcarboxamido)phenyl]ethynyl-3α-hydroxy-5α-pregnan-20-oneethyl ester (18 mg) as a colorless solid; TLC R_(f) (hexane:acetone3:1)=0.22.

EXAMPLE 16 3α-Hydroxy-3β-[5-oxo-1-hexynyl]-5β-pregnan-20-one cyclic(1,2-ethanediyl acetal)

A solution of 1-hexyn-5-one cyclic (1,2-ethanediyl acetal) (493 mg, 3.52mmol) in dry THF (15 mL) was treated with n-BuLi (2.5M in THF, 3 mmol,1.2 mL) at −60° C. After stirring the mixture at −78° C. for 0.5 hr, asolution of 5β-pregnan-3,20-dione, cyclic 20-(1,2-ethanediyl acetal)(360 mg, 1 mmol) in THF (15 mL) was added and the mixture was stirred at−78° C. for 1 hr. The cooling bath was removed and the mixture wasquenched with NH₄Cl solution (3 mL). The solvent was removed and theresidue was then dissolved in acetone (40 mL). After adding 1N HCl (4mL) the solution was stirred at rt for 15 min. Sat. NaHCO₃ soln. wasadded to neutralize the acid. The solvents were removed and the residuewas extracted with EtOAc. The organic layer was washed with water, dil.NaHCO₃ soln., water, and brine. After drying over anhyd. MgSO₄ thesolution was filtered and evaporated to yield the crude product (700mg). This crude product was then dissolved in a small amount of CH₂Cl₂and poured on a column of silica gel. Elution with toluene:acetonemixture (93:7) gave 3α-hydroxy-3β-[5-oxo-1-hexynyl]-5β-pregnan-20-onecyclic (1,2-ethanediyl acetal) (210 mg) as a colorless solid; mp130-133° C.; TLC R_(f) (toluene:acetone 9:1)=0.33.

EXAMPLE 17 3α-Hydroxy-3β-[5-oxo-1-hexynyl]-5β-pregnan-20-one

3α-Hydroxy-3β-[5-oxo-1-hexynyl]-5β-pregnan-20-one cyclic (1,2-ethanediylacetal) (95 mg) was dissolved in acetone (5 mL). After adding 2N HCl (1mL) the solution was stirred at rt for 3 hr. Sat. NaHCO₃ soln. was addedto neutralize the acid. The solvents were removed and the residue wasextracted with EtOAc. The organic layer was washed with water, water,and brine. After drying over anhyd. MgSO₄ the solution was filtered andevaporated to yield the crude title product (100 mg), which was thencrystallized from hexane-acetone as colorless rods, yield 63 mg; mp104-106° C.; TLC R_(f) (hexane:acetone 7:3)=0.27.

An analogous method was used to prepare3α-hydroxy-3β-[6-oxo-1-heptynyl]-5β-pregnan-20-one, using 1-heptyn-6-onecyclic 6-(1,2-ethanediyl acetal). PCS-TEXT

EXAMPLE 18 3β-(5-Cyano-1-pentynl)-3α-hydroxy-5β-pregnan-20-one

A solution of 5-cyanopentyne (0.84 mL, 8 mmol) in dry THF (20 mL) wastreated with n-BuLi (2.5 M in THF, 7.8 mmol, 3.2 mL) at −70° C. Afterstirring the mixture at −75° C. for 0.5 hr, a solution of5β-pregnan-3,20-dione, cyclic 20-(1,2-ethanediyl acetal) (720 mg, 2mmol) in THF (20 mL) was added and the mixture was stirred at −78° C.for 0.5 hr. The cooling bath was removed and the mixture was quenchedwith NH₄Cl solution (3 mL). The solvent was removed and the residue wasthen dissolved in acetone (40 mL). After adding 1N HCl (4 mL) thesolution was stirred at rt for 15 min. Sat. NaHCO₃ soln. was added toneutralize the acid. The solvents were removed and the residue wasextracted with EtOAc. The organic layer was washed with water, andbrine. After dying over anhyd. MgSO₄ solution was filtered andevaporated to yield the crude product (1.55 g). This crude product wasthen dissolved in a small amount of CH₂Cl₂ and poured on a column ofsilica gel. Elution with toluene:acetone mixture (95:5) gave3α(5-cyano-1-pentynyl)-3β-pregnan-20-one (170 mg) as a first fraction.Further elution with the same solvent mixture yielded3β-(5cyano-1-pentynyl)-3α-hydroxy-5β-pregnan-20-one (480 mg) as acolorless solid; mp 134-136° C.; TLC R_(f) (hexane:acetone 7:3)=0.3.

An analogous method was used to prepare3β-(4-cyano-1-butynyl)-3α-hydroxy-5β-pregnan-20-one.

EXAMPLE 19 3α-Hydroxy-3β-pyridyl)ethynyl-5β-pregnan-20-one

A solution of 2-ethynylpyridine (270 mg, 2.6 mmol) in dry THF (15 mL)was treated with a n-BuLi (2.5 M in THF, 2.5 mmol, 1 mL) at −60° C.After stirring the mixture at −78° C. for 0.5 hr, a solution of5β-pregnan-3,20-dione, cyclic 20-(1,2-ethanediyl acetal)170 mg, 047mmol) in THF (15 mL) was added and the mixture was stirred at −78° C.for 1 hr. The cooling bath was removed and the mixture was quenched withNH₄Cl solution (3 mL). The solvent was removed and the residue was thendissolved in acetone (25 mL). After adding 2N HCl (2 mL) the solutionwas stirred at rt for 15 min. Sat. NaHCO₃ soln. was added to neutralizethe acid. The solvents were removed and the residue was extracted withEtOAc. The organic layer was washed with water, and brine. After dryingover anhyd. MgSO₄ the solution was filtered and evaporated to yield thecrude product (360 mg). This crude product was then dessolved in a smallamount of CH₂Cl₂ and poured on a column of silica gel. Elution withtoluene:acetone mixture (95:5) gave the unreacted ethynlpyridine as afirst fraction. Further elution with the same solvent mixture yielded3β-(2-pyridyl)ethynyl-3α-hydroxy-5β-pregnan-20-one (107 mg) as acolorless solid; mp 192-195° C.; TLC R_(f) (toluene:acetone 87:13)=0.21.

EXAMPLE 20 3β-(6-hydroxy-1-hexynyl)-3α-hydroxy-5βpregnan-20-one

A solution of 5-hexyn-1-ol (1.35 mL, 12 mmol) in dry THF (15 mL) wastreated with n-BuLi (9.6 mL, 2.5 in THF, 24 mmol) at −65° C. Afterstirrig the mixture at −78° C. for 0.5 hr, a solution of5β-pregnan-3,20-dione, cyclic 20-(1,2-ethanediyl acetal)(1.08 g, 3 mmol)in THF (20 ml) was added and the mixture was stirred at −78° C. for 1hr. The cooling bath was removed and the stirring was continued at rtfor 45 min. The mixture was then quenched with NH4Cl solution (5 mL).The solvent was removed the residue was extracted with EtOAc. Theorganic layer was washed with water, dil. NaHCO₃ soln. water, and brine.After drying over anhyd. MgSO₄. The solution was filtered and evaporatedto yield the crude product (1.90 g). This crude product was thencrystallized from EtOAc to yield the pure product as colorless rods (890mg). This was then dissolved in acetone (120 mL). After adding 2N HCl (3mL) the solution was stirred at rt for 15 min. Sat. NaHCO₃ soln. wasadded to neutralize the acid. The solvents were removed and the residuewas extracted with EtOAc. The organic layer was washed with water, andbrine. After drying over anhyd. MgSO₄, the solution was filtered andevaporated to yield the crude product. This crude product was thendissolved in a small amount of CH₂Cl₂ and poured on a column of silicagel. Elution with tolene:acetone mixture (95:5) gave the unreactedhexynol as a first fraction. Further elution with the same solventmixture yielded 3α-(6-hydroxy-1-hexynyl)-3β-hydroxy-5β-pregnan-20-one(60 mg), and then 3β-(6-hydroxy-1-hexynyl)-3α-hydroxy-5-pregnan-20-one(620 mg) as a colorless solid; 132-134° C.; TLC R_(f) (hexane:acetone7:3)=0.23.

EXAMPLE 21 3β-(6′-Hydroxy-1′-hexynyl)3α-hydroxy-5β-pregnan-20-one6′-hemisuccinate sodium salt a.3β-(6′-Hydroxy-1′-hexanynyl)-3α-hydroxy-5β-pregnan-20-one6′-hemisuccinate

A solution of 3β-(6′-hydroxy-1′-hexynyl)-3α-hydroxy-5β-pregnan-20-one(600 mg, 1.45 mmol) in pyridine (5 mL) was treated with succinicanhydride (600 mg, 6 mmol) and 4-(N,N-dimethyl)aminopyridine (20 mg).The mixture was heated to 70-75° C. for 1.25 hr. The tlc showed 100%conversion. It was cooled to rt and was poured into ice-2N HCl. Theorganics was extracted with EtOAc. The organic layer was washed with0.2N HCl, water, and brine. After drying over anhyd. MgSO₄ the solutionwas filtered and evaporated to yield the crude product. This crudeproduct was then dissolved in a small mixture of CH₂Cl₂ and poured on acolumn of silica gel. Elution with hexane:acetone mixture (73) gave3α-(6′-hydroxy1-hexynyl)-3α-hydroxy-5β-pregnan-20-one 6′-hemisuccinate(700 mg); TLC R_(f) (hexane:acetone:AcOH 70:30:0.5)=0.21.

b. 3β-(6′-hydroxy-1′-hexynyl)-3α-hydroxy-5β-pregnan-20-one6′-hemisuccinate sodium salt

A mixture of the above hemisuccinate (400 mg), NaHCO₃ (68 mg), water (8mL), and CH₂Cl₂ (1 mL) was stirred at rt for 15 min. The solvent wasremoved and the residue was freeze-dried to yield the sodium salt as acolorless solid (400 mg).

EXAMPLE 22 3α-(5′-Hydroxy-1′-pentynyl)-3α-hydroxy-5α-pregnan-20-one

A solution of 4-pentyn-1-ol (1.1 mL, 12 mmol) in dry THF (15 mL) wastreated with n-BuLi (9.9 mL, 2.5 M in THF, 24.5 mmol) at —65° C. Afterstirring the mixture at −78° C. for 0.5 hr, a solution of5-β-pregnan-3,20-dione, cyclic 20-(1,2-ethanediyl acetal) (1.08 g, 3mmol) in THF (20 mL) was added and the mixture was stirred at −78° C.for 1 hr. The cooling bath was removed and the stirring was continued atrt for 45 min. The mixture was then quenched with NH₄Cl solution (5 mL).The solvent was removed the residue was then dissolved in acetone (30mL). After adding 2N HCl (7 mL) the solution was stirred at rt for 15min. Sat. NaHCO₃ soln. was added to neutralize the acid. The solventswere removed and the residue was extracted with EtOAc. The organic layerwas washed with water, and brine. After drying over anhyd. MgSO₄ thesolution was filtered and evaporated to yield the crude product. Thiscrude product was then dissolved in a small amount of CH₂Cl₂ and pouredon a column of silica gel. Elution with toluene:acetone:EtOAc mixture(70:15:15) gave the unreacted pentynol as a first fraction. Furtherelution with the same solvent mixture yielded3α-(5′-hydroxy-1′-pentynyl)-3β-hydroxy-5-pregnan-20-one (100 mg), andthen 3β-(5′-hydroxy-1′-pentynyl)-3α-hydroxy-5β-pregnan-20-one (650 mg)as a colorless solid: mp 160-163° C.; TLC R_(f) (toluene:acetone:EtOAc70:15:15)=028.

An analogus method was used toprepare:3β-(4′-hydroxy-1′-butynyl)-3α-hydroxy-5β-pregnan-20-one;3β-(4′-hydroxy-1′-butynyl)-3α-hydroxy-5β-19-nonpregnan-20-one;3β(4′-hydroxy-1′-butynyl)-3α-hydroxy-5α-pregnan-20-one; 3β-[4′(R/S)-hydroxy-1′-pentynyl]-3α-hydroxy-5α-pregnan-20-one;3β-(3′-hydroxy-1′-propynyl)-3α-hydroxy-5β-pregnan-20-one.

EXAMPLE 23 3β-(5′-Hydroxy-1′-pentynyl)-3α-hydroxy-5β-pregnan-20-one5′-hemissucinate sodium salt a.3β-(5′-Hydroxy-1′-pentynyl)3α-hydroxy-5β-pregnan-20-one 5′-hemisuccinate

A solution of 3β-(6′-hydroxy-1′-hexynyl)3α-hydroxy-5β-pregnan-20-one(350 mg, 0.87 mmol) in pyridine (3 mL) was treated with succinicanyhdride (380 mg, 3.8 mmol) and 4-(N,N-dimethyl)aminopyridine (20 mg).The mixture was heated to 65-70° C. for 1 hr. The TLC showed 100%conversion. It was cooled to rt and was poured into the ice-2N HCl. Theorganics were extracted with ETOAc. The organic layer was washed with0.2N HCl, water, and brine. After drying over anhyd. MgSO₄ the solutionwas filtered and evaporated to yield the crude product (900 mg). Thiscrude product was then dissolved in a small amount of CH₂Cl₂ and pouredon a column of silica gel. Elution with hexane:acetone mixture (7:3)gave 3β-(5′-hydroxy-1′-pentynyl)-3α-hydroxy-5β-pregnan-20-one5′-hemisuccinate (350 mg); TLC R_(f) (hexane:aceton:AcOH70:30:0.5)=0.25.

b. 3β-(5′-hydroxy1′-pentynyl)-3α-hydroxy-5β-pregnan-20-one5′-hemisuccinate sodium salt

A mixture of the above hemisuccinate (345 mg), NaHCO₃ (60 mg), water (5mL), THF (2 mL), and CH₂Cl₂ (1 mL) was stirred at rt for 1 hr. Thesolvent was removed and the residue was freeze-dried to yield the sodiumsalt as a colorless solid (340 mg).

An analogous method was used to prepare hemisuccinate sodium salts from:3β-(4′-hydroxy-1′-butynyl)-3α-hydroxy-5β-pregnan-20-one;3β-(4′-hydroxy-1′-butynyl)-3α-hydroxy-5β-19-nonpregnan-20-one;3β-(4′-hydroxy-1′-butynyl)-3α-hydroxy-5α-pregnan-20-one;3β-[3′(R/S)-hydroxy-1′-butynyl]-3α-hydroxy-5α-pregnan-20-one;and 3β-(3′-hydroxy-1′-propynyl)-3α-hydroxy-5β-pregnan-20-one.

EXAMPLE 24 3β-(5′-Acetoxy-1′-pentynyl)-3α-hydroxy-5β-pregnan-20-one

A solution of pyridine (280 mg) in CH₂Cl₂ (2 mL) was treated with acetylchloride (280 mg) at 0-5° C. A solution of3β-(5′-hydroxy-1′-pentynyl)-3α-hydroxy-5β-pregnan-20-one (130 mg) inCH₂Cl₂ (3 mL) was added. The stirring was continued at 0° C. for 20 min.The TLC showed 100% conversion, hence, the mixture was poured intoice-2N HCl (20 g, 2 mL). The organics were extracted with EtOAc. Theorganic layer was washed with 0.2N HCl, water, and brine. After dryingover anhyd. MgSO₄ the solution was filtered and evaporated to yield thecrude product. This crude product was then dissolved in a small amountof CH₂Cl₂ and poured on a column of silica gel. Elution withtoluene:acetone mixture(9:1)gave3β-(5′-acetoxy-1′-pentynyl)-3α-hydroxy-5β-pregnan-20-one (100 mg);mp 84-87° C.; TLC R_(f) (hexane:acetone 70:30)=0.38.

EXAMPLE 25 3β-(4′-Acetoxy-1′-butynyl)-3α-hydroxy-5β-pregnan-20-one

A solution of pyridine (280 mg) in CH₂Cl₂ (2 mL) was treated with acetylchloride (280 mg) at 10° C. A solution of3β-(4′-hydroxy-1′-butynyl)-3α-hydroxy-5β-pregnan-20-one (110 mg) inCH₂Cl₂ (3 mL) was added. The stirring was continued at 10° C. for 30min. The TLC showed 100% conversion, hence, the mixture was poured intoice-2N HCl (20 g, 3 mL). The organics were extraced with EtOAc. Theorganic layer was washed with 0.2N HCl, water, and brine. After dryingover anhyd. MgSO₄ the solution was filtered and evaporated to yield thecrude product. This crude product was then dissolved in a small amountof CH₂Cl₂ and poured on a column of silica gel. Elution withhexane:acetone mixture (4:1) gave3β-(4′-acetoxy-1′-butynyl)-3α-hydroxy-5β-pregnan-20-one (92 mg); mp170-173° C.; TLC R_(f) (hexane:acetone 70:30)=0.3.

EXAMPLE 26 3β-(6′-Aceotxy-1′-hexynyl)-3α-hydroxy-5β-pregnan-20-one

A solution of pyridine (2 mL) in Ch₂Cl₂ (10 mL) was treated with acetylchloride (2 mL) at 0° C. A solution of3β-(6′-hydroxy-1′-hexynyl)-3α-hydroxy-5β-pregnan-20-one (400 mg, 0.96mmol) in CH₂Cl₂ (5 mL) was added. The stirring was continued at 0° C.for 20 min. The TLC shoed 100% conversion, hence, the mixture was pouredinto ice-2N HCl (50 g, 11 mL). The organics wre extracted with EtOAc.The organic layer was washed with 0.2N HCl, water, and brine. Afterdying over anhyd. MgSO₄ the solution was filtered and evaporated toyield the crude product (500 mg). This crude product was then dissolvedin a small amount of CH₂Cl₂ and poured on a column of silica gel.Elution with toluene:acetone mixture (95:5)gave3β-(6′-acetoxy-1′-hexaynyl)-3α-hydroxy-5β-pregnan-20-one(130 mg); mp85-87° C.; TLC R_(f) (toluene:acetone 93:7)=0.2.

EXAMPLE 27 3α-Hydroxy-3β-[3-(2′-propnyloxy)-1-propynyl]5β-pregnan-20-one

A solution of propargyl ether (0.3 mL, 3 mmol) in dry THF (10 mL) wastreated with n-BuLi (2.4 M in THF, 3 mmol, 1.25 mL) at −70° C. Afterstirring the mixture at −75° C. for 0.5 hr, a solution of5β-pregnan-3,20-dione, cyclic 20-(1,2-ethanediyl acetal) (360 mg, 1mmol) in THF (10 mL) was added and the mixture was stirred at −78° C.for 1 hr. The cooling bath was removed an the mixture was quenched, withNH₄Cl solution (5 mL). The solvent was removed and the residue was thendissoved in acetone (40 mL). After adding 1N HCl (4 mL) the solution wasstirred at rt for 15 min. Sat NaHCO₃ soln. was added to neutralize theacid. The solvents were removed and the residue was extracted withEtOAc. The organic layer was washed with water, and brine. After dryingover anhyd. MgSO₄ the solution was filtered and evaporated to yield thecrude product. This crude product was then dissolved in a small amountof CH₂Cl₂ and poured on a column of silica gel. Elution withtoluene:acetone mixture (95:5) gave 3β-hydroxy-3β-[3-(2′-propynloxy)-1propynl]-5β-pregnan-20-one (31 mg) as a firstfraction. Further elution with the same solvent mixture yielded3α-hydroxy-3β- [3-(2′-propynyloxy)-1-propynyl]-5β-pregnan-20-one (255mg) as a colorless solid; mp 103-106° C,; TLC R_(f) (toluene:acetone955)=0.39

EXAMPLE 28 3α-Hydroxy-3β-(3-methoxy-1-propynyl)-5β-pregnan-20-one

A solution of methyl propargyl ether (0.25 mL, 3 mmol) in dry THF (10mL) was treated with a n-BuLi (2.4M in THF, 2.9 mmol, 1.20 mL) at −70°C. After stirring the mixture at −75° C. for 0.5 hr, a solution of5β-pregnan-3,20-dione, cyclic 20-(1,2-ethanediyl acetal) (185 mg, 0.5mmol) in THF (10 mL) was added and the mixture was stirred at −78° C.for 20 min. The cooling bath was removed and the mixture was quenchedwith NH₄Cl solution (2 mL). The solvent was removed and the residue wasthen dissolved in acetone (25 mL). After adding 2N HCl (2 mL) thesolution was stirred at rt for 15 min. Sat. NaHCO₃ soln. was added toneutralize the acid. The solvents were removed and the residue wasextracted with EtOAc. The organic layer was washed with water, andbrine. After drying over anhyd. MgSO₄ the solution was filtered andevaporated to yield the crude product (250 mg). This crude product wasthen dissolved in a small amount of CH₂Cl₂ and poured on a column ofsilica gel. Elution with toluene:acetone mixture (83:7) gave3β-hydroxy-3α-(3-methoxy-1-propnyl-5β-pregnan-20-one (19 mg) as a firstfraction. Further elution with the same solvent mixture yielded3α-hydroxy-3β-methoxy-1-propynyl)-5β-pregnan-20-one (115 mg) as acolorless solid; mp 155-159° C.; TLC R_(f) (hexane:acetone 7:3)=0.25.

EXAMPLE 293α-Hydroxy-3β-[3-(4′-pyridinyloxy)-1-propynyl]-5β-pregnan-20-one

A solution of propargyl 4-pyridyl ether (prepared according to (Thummelet al, J. Org. Chem. 1978, 43 4882) (173 mg, 1.3 mmol) in dry THF (15mL) was treated with n-BuLi (2.5M in THF, 1,3 mmol, 0.52 mL) at −70° C.After stirring the mixture at −75° C. for 0.5 hr, a solution of5β-pregnan-3,20-dione, cyclic 20-(1,2-ethanediyl acetal) (180 mg, 0.5mmol) in THF (15 mL) was added and the mixture was stirred at −78° C.for 20 min. The cooling bath was removed and the stirring was continuedat rt for 1 hr. The mixture was quenched with NH₄Cl solution (2 mL). Thesolvent was removed and the residue was then dissolved in acetone (25mL). After adding 2N HCl (2 mL) the s olution was stirred at rt for 20min. Sat. NaHCO₃ soln. was added to neutralize the acid. The solventswere removed and the residue was extracted with EtOAc. The organic layerwas washed with water, and brine. After drying over anhyd. MgSO₄ thesolution was filtered and evaporated to yield the crude product (250mg). This crude product was then dissolved in a small amount of CH₂Cl₂and poured on a column of silica gel. Elution with CH₂Cl₂:acetonemixture (90:10) gave3α-hydrocy-3β-[3-4′-pyridinyloxy)-1-propynyl]-5β-pregnan-20-one (145 mg)as a colorless solid; mp 84-90° C.; TLC R_(f) (CH₂Cl₂:acetone85:15)=0.17.

EXAMPLE 303α-Hydroxy-3β-[3-(1′H-1,2,3-triazol-1′-yl)-1-propynyl]-5β-pregnan-20-one

A solution of 1-(2-propynyl)-1H-1,2,3-triazole (prepared by the reactionof triazole with propargyl bromide) 77 mg, 0.72 mmol) in dry THF (10 mL)was treated with n-BuLi (2.5M in THF, 0.72 mmol, 0.28 mL) at −70° C.After stirring the mixture at −75° C. for 0.5 hr, a solution of5β-pregnan-3,20-dione, cyclic 20-(1,2-ethanediyl acetal) (130 mg, 0.36mmol) in THF (10 mL) was added and the mixture was stirred at −78° C.for 1 hr. The cooling bath was removed and the mixture was quenched withNH₄Cl solution (1 mL). The solvent was removed and the residue was thendissolved in acetone (25 mL). After adding 2N HCl (2 mL) the solutionwas stirred at rt for 30 min. Sat. NaHCO₃ soln. was added to neutralizethe acid. The solvents were removed and the residue was extracted withEtOAc. The organic layer was washed with water, and brine. After dryingover anhyd. MgSO₄ the solution was filtered and evaporated to yield thecrude product. This crude product was then dissolved in a small amountof CH₂Cl₂ and poured on a column of silica gel. Elution withtoluene:acetone mixture (85:15) gave3α-hydroxy-3β-[3-(1′H-1,2,3triazol-1′-yl)-1-propynyl]-5β-preggnan-20-one(56 mg) as a colorless solid; mp 142-144° C.; TLC R_(f) (hexane:acetone7:3)=0.33.

EXAMPLE 313αHydroxy-3β-[3-(2′H-1,2,3-triazol-2′-yl)-1-propynyl]-5β-pregnan-20-one

A solution of 2-(2-propynyl)-2H1,2,3-triazole (prepared by the reactionof triazole with propargyl bromide) (35 mg, 0.33 mmol) in dry THF (10mL) was treated with n-BuLi (2.5 in THF, 0.33 mmol, 0.15 mL) at −70° C.After stirring the mixture at −75° C. for 0.5 hr, a solution of5-pregnan-3,20-dione, cyclic 20-(1,2-ethanediyl acetal) (60 mg, 0.16mmol) in THF (10 mL) was added and the mixture was stirred at −78° C.for 1 hr. The cooling bath was removed and the mixture was quenched withNH₄Cl solution (1 mL). The solvent was removed and the residue was thendissolved in acetone (25 mL). After adding 2N HCl (2mL) the solution wasstirred at rt for 30 min. Sat. NaHCO₃ solsn. was added to neutralize theacid. The solvents were removed and the residue was extraced with EtOAc.The organic layer was washed with water, adn brine. After drying overanhyd. MgSO₄ the solution was filtered and evaporated to yield the crudeproduct. This curde product was then dissolved in a small amaount ofCH₂Cl₂ and poured on a column of silica gel. Elution withtoluene:acetone mixture (85:15) gave pregnan-3,20-dione (20 mg) as afirst fraction. Further elution with the same solvent yielded3α-hydroxy-3β-[3-(2′H-1,2,3-triazol-2′-yl)-1-proprnyl]-5β-pregnan-20-one(20-one (20 mg) as a colorless solid; mp139-140° C.; TLC R_(f)(hexane:acetone 4:1)=0.17.

EXAMPLE 32 3α-Hydroxy-3β- (2′-thienyl)ethynyl-5β-pregnan-20-one

A solution of 4iodothiophene (63 mg, 0.3 mmol),3β-ethynyl-3α-hydroxy-5β-pregnan-20-one (103 mg, 0.3 mmol) in drydegassed triethylamine (1 mL) was stirred under argon at 23° C.Bis(triphenylphosphine)palladium(II) dichloride (5 mg) and Cul (5 mg)were added and mixture was stirred at this temp. for 45 min. CH₂Cl₂ (5mL) was added and the mixture was stirred at 23° C. for 1 hr. The TLCshoed 100% conversion of the starting material, hence, the solvent wasremoved, and the residue was purfied by chromatography on silica gel.Elution with hexane:acetone (4:1) gave3α-hydroxy-3β-(2′thienyl)ethynyl-5β-pregnan-20-one (50 mg ) as acolorless solid; mp 205-206° C., TLC R_(f) (hexane:acetone 4:1)=0.35.

An analogus method was used to prepare3α-hydroxy-3β-(5′-acetyl-2′-thienyl) ethynyl-5β-pregnan-20-one; mp226-228 ° C., TLC R_(f) (hexane:acetone 4:1)=0.14.

EXAMPLE 33 3α-Hydroxy-3β-(3′-phenyl-1′-propynyl)-5βpregan-20-one

A solution of 3phenyl-1-propyne (0.25 mL, 2 mmol) in dry THF (17 mL) wastreated with n-BuLi (2.5M in THF, 2 mmol, 0.8 mL) at −70° C. Afterstirring the mixture at −75° C. for 10 min., a solution of5β-pregnan-3,20-dione, cyclic 20-(1,2-ethanediyl acetal) (206 mg, 0.6mmol) in THF (10 mL) was added and the mixture was stirred at −78° C.for 20 min. The cooling bath was removed and the mixture was quenchedwith NH₄Cl solution (5 mL). The solvent was removed and the residue wasthen dissolved in acetone (15 mL). After adding 2N HCI (4 mL) thesolution was stirred at rt for 20 min. Sat. NaHCO₃ soln. was added toneutralize the acid. The solvents were removed and the residue wasextracted with EtOAc. The organic layer was washed with water, andbrine. After drying over anhyd. MgSO₄ solution was filtered andevaporated to yield the crude product (460 mg). This crude product wasthen dissolved in a small amount of CH₂Cl₂ and poured on a column ofsilica gel. Elution with toluene:acetone mixture (85:15) gave theunreacted phenylpropyne as a first fraction. Further elution with thesame solvent yielded 3α-hydroxy-3β-(3′-propynyl)-5β-pregnan-20-one (175mg )as a colorless solid; mp 124-132° C.; TLC R_(f) (hexane:acetone7:3)=046.

EXAMPLE 34 3α-Hydroxy -3β-(3′-phenylpropyl)-5β-pregnan-20-one

A solution of the above phenylpropynyl derivative (50 mg) in EtOAc (10mL) was hydrogenated over Pd/C (10 mg, 5%) under 2 atm. of H₂ for 45min. The mixture was then filtered through a small pad of Celite, andconcentrated to yield the title compound as a colorless solid (40 mg);mp 41-46° C; R_(f) (hexane:acetone 7:3)=0.48.

EXAMPLE 35 3α-Hydroxy-3β- (3′phenylpropyl)-5β-pregnan-20-one

A solution of 1-(2-propynyl)-1H-pyrazole (prepared by the reaction ofpyrazole with propargyl bromide) (160 mg, 1.5 mmol) in dry THF (15 mL)was treated with n-BuLi (2.5M in THF, 1.5 mmol, 0.6 mL) at −70° C. Afterstirring the mixture at −75° C. for 0.5 hr, a solution of 5β-pregnan-3,20-dione, cyclic 20-(1.2-ethanediyl acetal) (180 mg, 0.5 mmol)in THF (15 mL) was added and the mixture was stirred at −78° C. for 1hr. The cooling bath was removed and the mixture was quenched with NH₄Clsolution (1 mL). The solvent was removed and the residue was thendissolved in acetone (25 mL). After adding 2N HCl (2 mL) the solution wsstirred at rt for 30 min. Sat. NaHCO₃ soln. was added to neutralize theacid. The solvents were removed and the residue was extracteed withEtOAc. The organic layer was washed with water, and brine. After dryingover anhyd. MgSO₄ the solution was filtered and evaporated to yield thecrude product. This crude product was then dissolved in a small amountof CH₂Cl₂ poured on a column of silicia gel. Elution withtoluene:acetone mixture (9:1) gave3α-hydroxy-3β-[3-(1′H-pyrazol-1′-yl)-1-propynyl]-5β-pregnan-20-one (80mg) as a colorless solid; mp 113-115° C. R_(f) (toluene:acetone9:1)=0.19.

EXAMPLE 36 3β-(3′-Acetylphenylenthynyl)-3α-hydroxy-5β-pregnan-20-one

A solution of 3-iodoacetophenone (74 mg, 0.3 mmol),3β-ethynyl-3α-hydroxy-5β-pregnan-20-one (103 mg, 0.3 mmol) in drydegassed triethylamine (1 mL) was stirred under argon at 23° C.Bid(triphenylphosphine)palladium(II) dichloride (5 mg) and Cul (5 mg)were added and the mixture was stirred at this temp. for 45 min. CH₂Cl₂(5 mL) was added and the mixture was stirred at 23° C. for 1.5 hr. TheTLC showed 100% conversion of the starting material, hence, the solventwas removed and the residue was purified by chromatography on silicagel. Elution with hexane:acetone (85:15) gave3β-(3′-acetylphenlethynyl)-3α-hydroxy-5β-pregnan-20-one (30 mg) as acolorless solid; mp ° C. TLC R_(f) (hexane:acetone 4:1)=0.

EXAMPLE 37 3β-(3′- Acetoxy-3′-propynyl)-3 α-hydroxy-5β-pregnan-20-one

A solution of pyridine (280 mg) in CH₂Cl₂ (2 mL) was treated with acetylchloride (280 ) at 0.5° C. A solution of3β-(3′-hydroxy-1′-propynyl)-3α-hydroxy-5β-pregnan-20-one (130 mg) inCH₂Cl₂ (3 mL) was added. The stirring was continued at 0° C. for 30 min.The TLC showed 100% conversion, hence, the mixture was poured intoice-2N HCl (20 g, 3 mL). The organics were extracted with EtOAc. Theorganic layer was washed with 0.2 N HCl, water, and brine. After dryingover anhyd. MgSO₄ the solution was filtered and evaporated to yield thecrude product (150 mg). This crude product was then dissolved in a smallamount of CH₂Cl₂ poured on a column of silica gel. Elution withtoluene:acetone mixture(4:1) gave3β-(3′-acetoxy-1′-propynyl)-3α-hydroxy-5β-pregnan-20-one (110 mg); mp132-150° C.; TLC r_(f) (hexane:acetone 70:30)=0.37.

EXAMPLE 383α-Hydroxy-3β-(4-hydroxybutyn-1-yl)-2(1-imidazolyl)-5β-pregnan-20-one a.2-Bromo-3α-hydroxy-3β-(4-hydroxbutyn-yl)-5β-pregnan-20-one

A solution of 3α-hydroxy-3β-(4-hydroxybutyn-1-yl)-5β-pregnan-20-one (386mg, 1 mmol) in MeOH (20 mL) was treated with two drops of acetylchloride, followed by bromine (1.2 mmol). The mixture was stirred at rtfor 2.5 hr and was poured into ice-water. The seperated solid wascollected by filtration, washed with water, dried (410 mg). Thissemi-dried solid was then dissolved in EtOAc and dried over anhyd.MgSO₄. Filtration and removal of the solvent gave the crude bromoderivative, and it was used as such for the next step.

b.3α-Hydroxy-3β-(4-hydroxybutyn-1-yl)-21-(1-imidazolyl)-5β-pregnan-20-one

A suspension of NaH (138 mg, 95%, 5.46 mmol) in THF (10 mL) was treatedwith a solution of imidazole (345 mg, 5.1 mmol) in THF (10 mL) at rt.After stirring the mixture for 30 min a solution of the crude bromoderivative (95 mg, 0.2 mmol) in THF (10 mL) was added. The stirring wascontinued at rt for 1 hr. Sat. NH₄Cl soln. was added and the solvent wasremoved. The residue was extracted with EtOAc. The organic layer waswashed with water, and brine. After drying over anhyd. MgSO₄ thesolution was filtered and evaporated to yield the crude product (100mg). This crude product was then dissolved in a small amount of CH₂Cl₂and poured on a column of silica gel. Elution with CH₂Cl₂:MeOH:NEt₃(95:4.5:0.5) mixture gave3α-hydroxy-3β-(4-hydroxybutyn-1-yl)-21-(1-imidazolyl-5β-pregnan-20-one(40 mg) as a colorless solid; mp 117-119° C.; TLC R_(f)(CH₂Cl₂:MeOH:NEt₃ 95:4.5:0.5) 0.21.

Similarly prepared were3α-hydroxy-3β-(4-hydroxybutyn-1-yl)-21-(1,2,4-triazol-1-yl)-5β-pregnan-20-one;mp 208-210° C.; TLC R_(f) (CH₂Cl₂:MeOH:NEt₃ 95:4.5:0.5) 0.24;3α-hydroxy-3β-(4-hydroxybutyn-1-yl)-21-(tetrazol-1-yl)-5β-pregnan-20-one;mp 110-112° C.; TLC R_(f) (CH₂Cl₂:MeOH:NEt₃ 96:3.5:0.5) 0.11;3α-hydroxy-3β-(4-hydroxybutyn-1-yl)-21-(1,2,3-triazol-1-yl)-5β-pregnan-20-one;mp 101-104° C.; TLC R_(f) (CH₂Cl₂:MeOH:NEt₃ 95:4.5:0.5) 0.2.

EXAMPLE 393α-Hydroxy-3β-(4-hydroxybutyn-1-yl)-21-(1-imidazolyl)-5β-19-norpregnan-20-onea. 21-Bromo-3α-hydroxy-3β-(4-hydroxybutyn-1-yl)-5β-19-norpregnan-20-one

A solution of3α-hydroxy-3β-(4-hydroxybutyn-1-yl)-5β-19-nonpregnan-20-one (710 mg. 1.9mmol) in MeOH (60 mL) was heated to 33° C. and was treated with twodrops of acetyl chloride, followed by bromine (2.28 mmol, 0.116 mL). Themixture was stirred at rt for 1.5 hr and was poured into ice-water. Theseparated solid was collected by filtration, washed with water, dried.This semi-dried solid was then dissolved in EtOAc and dried over anhyd.MgSO₄. Filtration and removal of the solvent gave the crude bromoderivative (870 mg), and it was used as such for the next step.

b.3α-Hydroxy-3β-(4-hydroxybutyn-1-yl)-21-(1-imidazolyl)-5β-19-nonpregnan-20-one

A suspension of NaH (138 mg, 95%, 5.46 mmol) in THF (10 mL) was treatedwith a solution of imidazole (345 mg, 5.1 mmol) in THF (10 mL) at rt.After stirring the mixture for 30 min a solution of the crude bromoderivative (90 mg, 0.2 mmol) in THF (10 mL) was added. The stirring wascontinued at rt for 1 hr. Sat. NH₄Cl soln. was added and the solvent wasremoved. The residue was extracted with EtOAc. The organic layer waswashed with water, and brine. After drying over anhyd. MgSO₄ thesolution was filtered and evaporated to yield the crude product (100mg). This crude product was then dissolved in a small amount of CH₂Cl₂and poured on a column of silica gel. Elution with CH₂Cl₂: MeOH:NEt₃(95:4.5:0.5) mixture gave3α-hydroxy-3β-(4-hydroxybutyn-1-yl)-21-(1-imidazolyl)-5β-19-norpregnan-20-one(25 mg) as a colorless solid; mp 118-127° C.; TLC R_(f)(CH₂Cl₂:MeOH:NEt₃ 95:4.5:0.5) 0.28.

EXAMPLE 403α-Hydroxy-3β-(4-hydroxybutyn-1-yl)-21-(1,2,3,triazol-2-yl)-5β-19-norpregnan-20one and3α-Hydroxy-3β-(4-hydroxybutyn-1-yl)-21-(1,2,3-triazol-1-yl)5β-19-norpregnan-20-one

A suspension of NaH (11 mg, 95%, 0.44 mmol) in THF (10 mL) was treatedwith a solution of 1,2,3-triazol (30 mg, 0.44 mmol) in THF (10 mL) atrt. After stirring the mixture for 30 min a solution of the crude21-bromo-3α-hydroxy-3β-(4-hydroxybutyn-1-yl)-5β-pregnan-20-one (100 mg,0.22 mmol) in THF (10 mL) was added. The stirring was continued at rtfor 1 hr. Sat. NH₄Cl soln. was added and the solvent was removed. Theresidue was extracted with EtOAc. The organic layer was washed withwater, and brine. After drying over anhyd. MgSO₄ the solution wasfiltered and evaporated to yield the crude product (100 mg). This crudeproduct was then dissolved in a small amount of CH₂Cl₂ and poured on acolumn of silica gel. Elution with CH₂Cl₂:acetone (4:1) mixture gave3α-hydroxy-3β-(4-hydroxybutyn-1-yl)-21-(2H-1,2,3-triazol-2-yl)-5β-19-norpregnan-20-one(15 mg) as a first fraction; mp 158-160° C.; TLC R_(f) (CH₂Cl₂:acetone4:1) 0.36 . Further elution with the same solvent mixture yielded3α-hydroxy-3β-(4-hydroxybutyn-1-yl)-21-(1H-1,2,3-triazol-1-yl)-5β-19-norpregnan-20-one(15 mg); 188-190° C.; TLC R_(f) (CH₂Cl₂:acetone 4:1) 0.2.

EXAMPLE 413α-Hydroxy-2β-morpholinyl-21-[1′,2′,4′-triazolyl)]-5α-pregnan-20-one

To a solution of 1,2,4-triazole (108 mg, 1.57 mmol) in dry THF (2.5 ml)was added sodium hydride (36 mg, 1.50 mmol). The mixture was stirred at25° C. under argon for 0.5 hour. Then3α-hydroxy-2β-morpholinyl-21-bromo-5α-pregnan-20-one (130 mg, 0.27 mmol)was added and the mixture was further stirred at 25° C. under argon for2.5 hours. Water (20 ml) was added slowly to the reaction mixture. Theproduct was extracted with ethyl acetate (3×25 ml). The combinedextracts were washed with brine (20 ml) and water (3×20 ml). The organicsolution obtained was dried over sodium sulfate and the solvent wasremoved in vacuo to give the crude product (130 mg). The pure product(90 mg, 75%) was obtained by flash chromatography 30 g of silica gel and300 ml of mixed solvent of CH₂Cl₂:MeOH: Et₃N=95:4.5:0.5, R_(f) =0.14).

EXAMPLE 42 3α-Hydroxy-21-(1′-uracil)-5α-pregnan-20-one

To a suspension of uracil (112 mg, 1 mmol) and K₂CO₃ (138 mg, 1mmol) inDMF (1.5 mL) was added 3α-hydroxy-21-bromo-5α-pregnan-20-one (400 mg, 1mmol) and the mixture obtained was stirred at 25° C. for 65 h. It wasthen poured into a separatory funnel containing water (60 ml) and ethylacetate (100 mL). The water layer was removed after shaking and thewhite solid in the organic layer was obtained by filtration as theproduct (87 mg, 20%). m.p.:262-265° C. (decomp).

EXAMPLE 43 Sodium S-(3α-hydroxy-5α-pregnan-20-on-21-yl) thiosulfate

To a solution of 21-bromo-3α-hydroxy-5α-pregnan-20-one (500 mg, 1.26mmol) dissolved in 50 mL of ethanol was added dropwise a solution ofsodium thiosulfate (199 mg, 1.26 mmol) in 10 mL of water. The reactionwas allowed to stir at rt until TLC (1:1 acetone/dichloromethane)indicated complete consumption of the starting bromide. Concentration ofthe reaction in vacuo gave the desired thiosulfate contaminated withsodium bromide. Flash column chromatography (silica gel eluted with agradient from 1:1 to 3:1 acetone/dichloromethane) afforded 430 mg (75%)of the title compound as a white solid.

Similarly prepared weresodiumS-(3α-hydroxy-3β-methyl-5α-pregnan-20-on-21-yl) thiosulfate,purified by flash chromatography as above, sodiumS-(3α-hydroxy-3β-methoxymethyl-5α-pregnan-20-on-21-yl) thiosulfate,purified by recrystallization from 50:1 methanol/water, sodiumS-(3α-hydroxy-5β-pregnan-20-on-21-yl) thiosulfate, purified by flashchromatography with silica gel and 3:1 acetone/ethanol, sodiumS-[3α-hydroxy-3β-(4′-hydroxybutynyl)-5β-pregnan-20-on-21-yl]thiosulfate, purified by trituration with hot acetone, andsodiumS-(3α-hydroxy-3β-trifluoromethyl-5β-19-norpregnan-20-on-21-yl)thiosulfate, purified by trituration with hot acetone/chloroform.

EXAMPLE 44 3α-Hydroxy-3β-(E)-(2-phenylethenyl)-5α-pregnan-20-one

Benzyl phenyl sulfoxide

To a solution of benzyl phenyl sulfide (Aldrich; 1.068 g, 5.33 mmol) in25 mL of CH₂Cl₂ at −78° C. was slowly added a solution ofm-chloroperbenzoic acid (Aldrich, 50-60%; 760 mg, 2.64 mmol if 60%) in10 mL of CH₂CL₂. After warming to room temperature and stirringovernight, the solution was added to 20 mL of a saturated NaHCO₃solution. The aqueous layer was separated and extracted with CH₂Cl₂(2×10 mL). The pooled organic layers were then dried (MgSO₄) andconcentrated. The residue was subjected to flash column chromatography(silica gel, 10% acetone/hexane and 15% acetone/hexane) affording thesulfoxide (632 mg, 55%) as a white solid, mp 123-126° C.

20,20-ethylenedioxy-3α-hydroxy-3β-[[2-(phenylsulfinyl)-2-phenyl]ethyl]-5α-pregnane

A solution of diisopropylamine (Aldrich, freshly distilled fromCaH_(2; 0.5) mL, 361 mg, 3.57 mmol) in 2 mL of dry THF was cooled to−10° C. and treated with a 1.6M solution of n-BuLi in hexanes (Aldrich;1.0 mL, 1.6 mmol) added dropwise via syringe. After 10 m, the reactionwas cooled to −75° C. and a solution of benzyl phenyl sulfoxide (347 mg,1.60 mmol) in 5 mL of dry THF was added dropwise via syringe over 30 m.To the resulting deep yellow solution was added 297 mg (0.79 mmol) ofsolid 20,20-ethylenedioxy-3(R)-5α-pregnan-3-spiro-2′-oxirane. Thereaction was allowed to warm to rt and then warmed to 50° C. After 5 h,the reaction was allowed to cool to rt and added to 30 mL of ice-coldwater. The resulting mixture was extracted with EtOAc (3×20 mL). Thecombined EtOAc layers were back extracted with a sat. NaCl solution,dried (Na₂SO₄) and concentrated. The residue was purified by flashchromatography (silica gel, gradient from 100% CH₂Cl₂ to 20%acetone/CH₂Cl₂.) affording the sulfoxide (405 mg, 86%) as a mixture oftwo diastereomers (based on two benzylic protons in ¹H NMR).

3α-Hydroxy-3β-[[2-(phenylsulfinyl)-2-phenyl]ethyl]-5α-pregnan-20-one

A solution of20,20-ethylenedioxy-3α-hydroxy-3β-[[2-(phenylsulfinyl)-2-phenyl]ethyl]-5α-pregnane(1.30 g, 2.21 mmol) in 15 mL of THF was cooled in an ice/water bath andan aqueous 1N HCl solution (3 mL) was added followed by 2 mL of acetone.After stirring at rt for 70 min, the reaction was recooled to 0° C. andadded to an EtOAc/water mixture containing 6 mL of a sat. NaHCO₃solution. The aqueous layer was extracted twice with EtOAc and thecombined organic layers were extracted with a sat. NaCl solution, dried(Na₂SO₄) and concentrated. The residue was carried on to the next stepwithout purification.

3α-Hydroxy-3β-(E)-(2-phenylethenyl)-5α-pregnan-20-one

A suspension of3α-hydroxy-3β-[[2-(phenylsulfinyl)-2-phenyl]ethyl]-5α-pregnan-20-one(963 mg, 1.76 mmol) in 6 mL of p-cymene containing 0.95 mL of2,4,6-collidine was heated at 135° C. of 1 hr. After standing overnightat 0° C., the product had precipitated and was isolated by filtration.The crude product was washed with cold p-cymene (3×5 mL) and hexane (atotal of 120 mL) and afforded 445 mg of the title compound as a whitesolid, mp 209-215° C.

EXAMPLE 45 3α-Hydroxy-2β-trimethylsilylethynyl-5α-pregnan-20-one

A solution of trimethylsilylacetylene (0.5 mL, 0.347 g, 3.54 mmol) in 4mL of dry THF was cooled to −78° C. and treated with a 2.5M solution ofn-BuLi in hexanes. Neat BF₃-ET₂O (0.3 mL, 0.346 g, 2.44 mmol) was addedvia syringe. After stirring at −78° C. of 5 m, a solution of20,20-ethylenedioxy-5α-pregnan-2α,3α-epoxide (557 mg, 1.54 mmol) in 3 mLof dry THF was added over 12 m. After an additional 2.5 h, the reactionwas added to a sat. NH₄Cl solution. The resulting mixture was extractedwith EtOAc (3×25 mL). The combined organic layers were washed with abrine solution, dried (Na₂SO₄) and concentrated under reduced pressure.The crude reaction mixture was dissolved in 10 mL of acetone, cooled inan ice/water bath and treated with a 1N HCl solution (1 mL). After 1 hat rt, the reaction was added to an ether/water mixture containing 1 mLof a sat. NaHCO₃ solution. The aqueous layer was washed twice with etherand the pooled ether layers were washed with a sat. NaCl solution, dried(Na₂SO₄) and concentrated. Flash column chromatography (silica gel,12.5%acetone/hexane) afforded 249 mg of the title compound as a white solid,mp 164-167° C.

The procedure above was used for the preparation of3α-hydroxy-2β-(3′-methoxy-1′-propynyl)-5α-pregnan-20-one.

The hydrolysis of the trimethylsilyl compound with K₂CO₃/MeOH gave3α-hydroxy-2β-ethynyl-5α-pregnan-20-one as a white solid, m.p. 179-189°C. (decomp.).

EXAMPLE 46 3α-21-Dihydroxy-3β-ethynyl-5β-pregnan-20-one a.21-Bromo-3β-ethynyl-3α-hydroxy-5β-pregnan-20-one

A solution of 3β-ethynyl-3α-hydroxy-5β-pregnan-20-one 3 g, 8.77 mmol) inMeOH (110 mL) was treated with two drops of HBr (48%), followed bybromine (0.5 mL, 10.8 mmol). The mixture was stirred at room temperaturefor 1 hr and was poured into ice-water. The separated solid wascollected by filtration, washed with water, and dried (3.2 g). Thissemi-dried solid was then dissolved in EtOAc and dried over anhyd.MgSO₄. Filtration and removal of the solvent gave the crude bromoderivative. This crude product was then dissolved in a small amount ofCH₂Cl₂ and poured on a column of silica gel. Elution with hexane:EtOAcmixture (4:1) gave 21-bromo-3β-ethynyl-3α-hydroxy-5β-pregnan-20-one (3.2g).

b. 3α,21-Dihydroxy-3β-ethynyl-5β-pregnan-20-one

A solution of the above bromo derivative (3.2 g) in acetone (100 mL) wastreated with trifluoroacetic acid (5.8 mL, 76 mmol) and triethylamine(8.5 mL). The mixture was refluxed for 30 min. and then trifluoroaceticacid sodium salt (10 g) was added in portions over a period of 10 hr.After cooling to room temperature, a solution of sat. NaHCO₃ was added.The solvent was removed and the residue was extracted with EtOAc. Theorganic layer was washed with water, and brine. After drying over anhyd.MgSo₄ the solution was filtered and evaporated to yield the crudeproduct. This crude product was then dissolved in a small amount ofCH₂Cl₂ and poured on a column of silica gel. Elution with hexane:EtOAcmixture (3:2) gave 3α,21-dihydroxy-3β-ethynyl-5β-pregnan-20-one (1.6 g).mp 156-157° C.; TLC R_(f) (hexane:acetone 3:1)=0.2.

EXAMPLE 47 3β-(4′-Acetylphenylethynyl)-3α,21-dihydroxy-5β-pregnan-20-one

A solution of 4-iodoacetophenone (1.22 g, 4.44 mmol),3α,21-dihydroxy-3β-ethynyl-5β-pregnan-20-one (1.59 g, 4.44 mmol) in drydegassed triethylamine (7 mL) was stirred under argon at 23° C.Bis(triphenylphosphine)palladium chloride (15 mg) and CuI (5 mg) wereadded and the mixture was stirred at this temp. for 45 min. CH₂Cl₂ (30mL) was added and the mixture was stirred at 23° C. for 1.5 hr. TLCshowed 100% conversion of the starting material, hence, the solvent wasremoved and the residue was purified by chromatography on silica gel.Elution with hexane:acetone (4:1) gave3β-(4′-acetylphenylethynyl)3α,21-dihydroxy-5β-pregnan-20-one (1.3 g) asa colorless solid; mp 181-183° C., TLC R_(f) (hexane:acetone 3:2)=0.14.

EXAMPLE 48 3β-(4′-Acetylphenylethynyl)-3α,21-dihydroxy-5β-pregnan-20-one21-hemisuccinate sodium salt a.3β-(4′-Acetylphenylethynyl)-3α,21-dihydroxy-5β-pregnan-20-one21-hemisuccinate

A solution of 3β-(4′-acetylphenylethynyl)-3α,21-dihydroxy-5β-pregnan-20-one (1.3 g, 6.76 mmol) in pyridine (8 mL) wastreated with succinic anhydride (680 mg, 2.5 mmol) and4-(N,N-dimethyl)aminopyridine (25 mg). The mixture was heated to 70-75°C. for 2.5 hr. TLC showed 100% conversion. Pyridine was removed and theresidue was extracted with EtOAc. The organic layer was washed with 0.1NHCl, water, and brine. After drying over anhyd. MgSO₄, the solution wasfiltered and evaporated to yield the crude product. This crude productwas then dissolved in a small amount of CH₂Cl₂ and poured on a column ofsilica gel. Elution with hexane:acetone mixture (7:3) gave3β-(4′-acetylphenyl-ethynyl)-3α,21-dihydroxy-5β-pregnan-20-one21-hemisuccinate (1.4 g).

b.3β-(4′-Acetylphenylethynyl)-3α,21-dihydroxy-5β-pregnan-20-one21-hemisuccinatesodium salt

A mixture of the above hemisuccinate (1.5 g, 2.6 mmol), NaHCO₃ (220 mg,2.6 mmol), water (10 mL), and CH₂Cl₂ (16 mL) was stirred at roomtemperature for 1 hr. The solvent was removed and the residue wasfreeze-dried to yield the sodium salt as a colorless solid (1.4 g).

EXAMPLE 49 3α,21-Dihydroxy-3β-ethynyl-5α-pregnan-20-one a.21-Bromo-3β-ethynyl-3α-hydroxy-5α-pregnan-20-one

A solution of 3β-ethynyl-3α-hydroxy-5α-pregnan-20-one (2.3 g, 6.72 mmol)in MeOH (80 mL) was treated with two drops of HBr (48%), followed bybromine (0.4 mL, 7.73 mmol). The mixture was stirred at room temperaturefor 1 hr and was poured into ice-water. The separated solid wascollected by filtration, washed with water, and dried (2.5 g). Thissemi-dried solid was then dissolved in EtOAc and dried over anhyd.MgSO₄. Filtration and removal of the solvent gave the crude bromoderivative. This crude product was then dissolved in a small amount ofCH₂Cl₂ and poured on a column of silica gel. Elution with hexane:EtOACmixture (85:15) gave 21-bromo-3β-ethynyl-3α-pregnan-20-one 2.1 g).

b. 3α,21-Dihydroxy-3β-ethynyl-5α-pregnan-20-one

A solution of the above bromo derivative (2.1 g, 5 mmol) in acetone (40mL) was treated with trifluoroacetic acid (3.8 mL, 50 mmol) andtriethylamine (5.5 mL). The mixture was refluxed for 30 min. and thentrifluoroacetic acid sodium salt (10 g) was added in parts over a periodof 10 hr. After cooling to room temperature, a solution of sat. NaHCO₃was added. The solvent was removed and the residue was extracted withEtOAc. The organic layer was washed with water, and brine. After dryingover anhyd. MgSO₄ the solution was filtered and evaporated to yield thecrude product (1.6 g). This crude product was then dissolved in a smallamount of CH₂Cl₂ and poured on a column of silica gel. Elution withhexane:EtOAc mixture (7:3) gave3α,21-dihydroxy-3β-ethynyl-5α-pregnan-20-one (1.5 g), mp 214-218° C.;TLC R_(f) (hexane:acetone 7:3)=0.33.

EXAMPLE 50 3β-(4′-Acetylphenylethynyl-3α,21-dihydroxy-5α-pregnan-20-one

A solution of 4-iodoacetophenone (962 mg, 4 mmol),3α,21-dihydroxy-3β-ethynyl-5α-pregnan-20-one (1.2 g, 4 mmol) in drydegassed triethylamine (7 mL) was stirred under argon at 23° C.Bis(triphenyl-phosphine)palladium chloride (15 mg) and CuI (5 mg) wereadded and the mixture was stirred at this temp. for 45 min. CH₂Cl₂ (25mL) was added and the mixture was stirred at 23° C. for 1.5 hr. TLCshowed 100% conversion of the starting material, hence, the solvent wasremoved and the residue was purified by chromatography on silica gel.Elution with hexane:acetone (7:3) gave3β-(4′-acetylphenylethynyl)-3α,21-dihydroxy-5α-pregnan-20-one (750 mg)as a colorless solid; mp 222-225° C., TLC R_(f) (hexane:acetone4:1)=0.13.

EXAMPLE 51 3β-(4′-Acetylphenylethynyl)-3α,21-dihydroxy-5α-pregnan-20-one21-hemisuccinate sodium salt a.3β-(4′-Acetylphenylethynyl)-3α,21-dihydroxy-5α-pregnan-20-one21-hemisuccinate

A solution of3β-(4′-acetylphenylethynyl)-3α,21-dihydroxy-5α-pregnan-20-one (276 mg,1.4 mmol) in pyridine (4 mL) was treated with succinic anhydride (680mg, 2.7 mmol) and 4-(N,N-dimethyl)aminopyridine (20 mg). The mixture washeated to 70-75° C. for 2.5 hr. TLC showed 100% conversion. Pyridine wasremoved in vacuo and the residue was extracted with EtOAc. The organiclayer was washed with 0.1N HCl, water, and brine. After drying overanhyd. MgSO₄ the solution was filtered and evaporated to yield the crudeproduct. This crude product was then dissolved in a small amount ofCH₂Cl₂ and poured on a column of silica gel. Elution with hexane:acetonemixture (7:3) gave3β-(4′-acetylphenylethynyl)-3α,21-dihydroxy-5α-pregnan-20-one21-hemisuccinate (400 mg).

b.3β-(4′-Acetylphenylethynyl)-3α,21-dihydroxy-5α-pregnan-20-one21-hemisuccinatesodium salt

A mixture of the above hemisuccinate (400 mg, 0.7 mmol), NaHCO₃ (60 mg,0.7 mmol), water (3mL), and Ch₂Cl₂ (5 mL) was stirred at roomtemperature for 1 hr. The solvent was removed and the residue wasfreeze-dried to yield the sodium salt as a colorless solid (390 mg).

EXAMPLE 52 3α-Hydroxy-3β-(3-pyridyl)ethynyl-5β-pregnan-20-one

A solution of 3-bromopyridine (70 mg, 0.44 mmol),3β-ethynyl-3α-hydroxy-5α-pregnan-20-one (150 mg, 0.44 mmol) in drydegassed diethylamine (1 mL) was stirred under-argon at 23° C.Bis(triphenyl-phosphine)palladium chloride (10 mg) and CuI (5 mg) wereadded and the mixture was stirred at this temp. for 45 min. CH₂Cl₂ (4mL) was added and the mixture was stirred at 23° C. for 1.5 hr. TLCshowed 100% conversion of the starting material, hence, the solvent wasremoved and the residue was purified by chromatography on silica gel.Elution with hexane:acetone (3:1) gave3α-hydroxy-3β-(3-pyridyl)ethynyl-5β-pregnan-20-one (30 mg) as acolorless solid; mp 230-35° C., TLC R_(f) (hexane:acetone 7:3)=0.24.

EXAMPLE 5321-Bromo-3α-hydroxy-3β-(3-hydroxypropyn-1-yl)-5β-pregnan-20-one

A solution of 3α-hydroxy-3β-(3-hydroxypropyn-1-yl)-5β-pregnan-20-one(483 mg, 1.3 mmol) in MeOH (20 mL) was heated to 33° C. and was treatedwith two drops of HBr (48%), followed by bromine (1.43 mmol, 0.73 mL).The mixture was stirred at 37° C. for 1 hr and was poured intoice-water. The separated solid was collected by filtration, washed withwater, and dried. This semi-dried solid was then dissolved in EtOAc anddried over anhyd. MgSO₄. Filtration and removal of the solvent gave thecrude 21-bromo-3α-hydroxy-3β-(3-hydroxypropyn-1-yl)-5β-pregnan-20-one(322 mg).

EXAMPLE 543α-Hydroxy-3β-(3-hydroxypropyn-1-yl)-21-(1,2,3-triazol-2-yl)-5β-pregnan-20-oneand 3α-hydroxy-3β-(3-hydroxypropyn-1-yl)-21-(1,2,3-triazol-1-yl)-5β-pregnan-20-one

A suspension of NaH (30mg, 95%, 1.2 mmol) in THF (10 mL) was treatedwith a solution of 1,2,3-triazole (69 mg, 1 mmol) in THF (10 mL) at roomtemperature. After stirring the mixture of 30 min. a solution of thecrude bromo derivative of Example 53 (162 mg, 0.37 mmol) in THF (10 mL)was added. The stirring was continued at room temperature for 1.5 hr.Sat. NH₄Cl soln. was added and the solvent was removed. The residue wasextracted with EtOAc. The organic layer was washed with water, andbrine. After drying over anhyd. MgSO₄ the solution was filtered andevaporated to yield the crude product (250 mg). This crude product wasthen dissolved in a small amount of CH₂Cl₂ and poured on a column ofsilica gel. Elution with CH₂Cl₂:acetone (6:4) mixture gave3α-hydroxy-3β-(3-hydroxypropyn-1-yl)-21-(2H-1,2,3-triazol-2yl)-5β-pregnan-20-one(40 mg) as a first fraction; mp 188-191° C.; TLC R_(f) (CH₂Cl₂:acetone6:4) 0.56. Further elution with the same solvent mixture yielded3α-hydroxy-3β-(3-hydroxypropyn-1-yl)-21-(1H-1,2,3-triazol-1-yl)-5β-pregnan-20-one(40 mg); mp 135-140° C.; TLC R_(f) (CH₂Cl₂:acetone 6:4) 0.34.

EXAMPLE 55 3β-(4′-Hydroxy-1′-butynyl)-3α-hydroxy-5β-pregnan-11,20-dione

A solution of 3-butyn-1-ol (0.4 mL, 5.2 mmol) in dry THF (15 mL) wastreated with n-BuLi (4 mL, 2.5M in THF, 10 mmol) at −65° C. Afterstirring the mixture at −78° C. for 0.5 hr, a solution of5β-pregnan-3,11,20-trione, cyclic 20-(1,2-ethanediyl acetal) (800 mg,2.14 mmol) in THF (20 mL) was added and the mixture was stirred at −78°C. for 1 hr. The cooling bath was removed and the stirring was continuedat room temperature for 45 min. The mixture was then quenched with NH₄Clsolution (5 mL). The solvent was removed the residue was then dissolvedin acetone (30 mL). After adding 2N HCl (7 mL) the solution was stirredat rt for 15 min. Sat. NaHCO₃ soln. was added to neutralize the acid.The solvents were removed and the residue was extracted with EtOAc. Theorganic layer was washed with water, and brine. After drying over anhyd.MgSO₄ the solution was filtered and evaporated to yield the crudeproduct. This crude product was then dissolved in a small amount ofCH₂Cl₂ and poured on a column of silica gel. Elution withtoulene:acetone mixture (3:1) gave the unreacted butynol as a firstfraction. Further elution with the same solvent mixture yielded3α-(4′-hydroxy-1′-butynyl)-3β-hydroxy-5β-pregnan-11,20-dione (100 mg),and then 3β-(4′-hydroxy-1′-butynyl)-3α-hydroxy-5β-pregnan-11,20-dione(400 mg) as a colorless solid; mp 158-162° C.; TLC R_(f)(toluene:acetone 3:1)=0.16.

It will be obvious to one skilled in the art that the above describedcompounds may be present as mixtures of diastereomers which may beseparated into individual diastereomers. Resolution of the diastereomersmay be conveniently accomplished by gas or liquid chromatography orisolation from natural sources. Unless otherwise specified herein,reference in the specification and claims to the compounds of theinvention, as discussed above, is intended to include all isomers,whether separated or mixtures thereof.

Where isomers are separated, the desired pharmacological activity willoften predominate in one of the diastereomers. As disclosed herein,these compounds display a high degree of stereospecificity. Inparticular, those compounds having the greatest affinity for the GABAreceptor complex are those with 3β-substituted-3α-hydroxypregnanesteroid skeletons.

The compounds of and used in the invention, that being the nontoxic,pharmaceutically acceptable, natural and synthetic, direct acting and“prodrug” forms of progesterone, deoxycorticosterone, and androstanemetabolites, have hitherto unknown activity in the brain at the GABA_(A)receptor complex. The present invention takes advantage of the discoveryof this previously unknown mechanism and activity.

The pharmaceutical compositions of this invention are prepared inconventional dosage unit forms by incorporating an active compound ofthe invention or a mixture of such compounds, with a nontoxicpharmaceutical carrier according to accepted procedures in a nontoxicamount sufficient to produce the desired pharmacodynamic activity in asubject, animal or human. Preferably, the composition contains theactive ingredient in an active, but nontoxic amount, selected from 1 mgto about 500 mg of active ingredient per dosage unit. This quantitydepends on the specific biological activity desired and the condition ofthe patient. Desirable objects of the compositions and methods of thisinvention are in the treatment of stress, anxiety, PMS, PND, andseizures such as those caused by epilepsy to ameliorate or prevent theattacks of anxiety, muscle tension, and depression common with patientssuffering from these central nervous system abnormalities. An additionaldesirable object of the composition and methods is to treat insomnia andproduce hypnotic activity. Another desirable object of the compounds anmethods is to induce anesthesia, particularly by intravenousadministration.

The pharmaceutical carrier employed may be, for example, either a solid,liquid, or time release (see e.g. Remington's Pharmaceutical Sciences,14th Edition, 1970). Representative solid carriers are lactose, terraalba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate,stearic acid, microcrystalline cellulose, polymer hydrogels and thelike. Typical liquid carriers are propylene glycol, glycofurol, aqueoussolutions of cyclodextrins, syrup, peanut oil, and olive oil and thelike emulsions. Similarly, the carrier or diluent may include anytime-delay material well known to the art, such as glycerol monostearateor glycerol distearate alone or with wax, microcapsules, microspheres,liposomes, and/or hydrogels.

A wide variety of pharmaceutical forms can be employed. Thus, when usinga solid carrier, the preparation can be plain milled micronized, in oil,tableted, placed in a hard gelatin or enteric-coated capsule inmicronized powder or pellet form, or in the form of a troche or lozenge.The compounds of and used in the invention may also be administered inthe form of suppositories for rectal administration. Compounds may bemixed in material such as cocoa butter and polyethylene glycols or othersuitable non-irritating material which is solid in rt but liquid at therectal temperature. When using a liquid carrier, the preparation can bein the form of a liquid, such as an ampule, or as an aqueous ornonaqueous liquid suspension. Liquid dosage forms also needpharmaceutically acceptable preservatives and the like. In addition,because of the low doses that will be required as based on the datadisclosed herein, parental administration, nasal spray, sublingual andbuccal administration, and timed release skin patches are also suitablepharmaceutical forms for topical administration.

The method of producing anxiolytic, anticonvulsant, mood altering (suchas anti-depressant) or hypnotic activity, in accordance with thisinvention, comprises administering to a subject in need of such activitya compound of the invention, usually prepared in a composition asdescribed above with a pharmaceutical carrier, in a nontoxic amountsufficient to produce said activity.

During menses, the levels of excreted metabolites vary approximatelyfourfold (Rosciszewska, et al.). Therefore, therapy for controllingsymptoms involves maintaining the patient at a higher level ofprogesterone metabolites than normal in the premenstrual state of PMSpatients. Plasma levels of active and major metabolites are monitoredduring pre-menses and post-menses of the patient. The amount of thecompounds of the invention administered, either singly or as mixturesthereof are thus calculated to reach a level which will exertGABA_(A)-receptor activity equal or higher than the level ofprogesterone metabolites in the normal subject during the premensesstate.

The route of administration may be any route that effectively transportsthe active compound to the GABA_(A) receptors that are to be stimulated.Administration may be carried out parenterally, enterally, rectally,intravaginally, intradermally, intramuscularly, sublingually, ornasally; the oral, intramuscular, and dermal routes are preferred. Forexample, one dose in a skin patch may supply the active ingredient tothe patient for a period of up to one week. However, the parenteralroute is preferred for status epilepticus.

Potency and Efficacy at the GR Site

The in vitro and in vivo experimental data show that thenaturally-occurring metabolites of progesterone/deoxycorticosterone andtheir derivatives interact with high affinity at a novel and specificrecognition site on the GR complex to facilitate the conductance ofchloride ions across neuronal membranes sensitive to GABA (Gee et al,1987 Harrison et al., 1987).

To those skilled in the art, it is known that the modulation of[³⁵S]t-butylbicyclophosphorothionate ([³⁵S]TBPS) binding is a measure ofthe potency and efficacy of drugs acting at the GR complex, which drugsmay be of potential therapeutic value in the treatment of stress,anxiety, and seizure disorders (Squires, R. F., et al., “[³⁵S]t-Butylbicyclophophorothionate binds with high affinity tobrain-specific sites coupled to a gamma aminobutyric acid-A and ionrecognition site,” Mol, Pharmacol., 23:326, 1983; Lawrence, L. J., etal., “Benzodiazepine anticonvulsant action: gamma-aminobutyricacid-dependent modulation of the chloride ionophore,” Biochem. Biophys.Res. Comm., 123:1130-1137, 1984; Wood, et al., “In vitrocharacterization of benzodiazepine receptor agonists, antagonists,inverse agonists and agonist/antagonists,” Pharmacol, Exp. Ther.,231:572-576, 1984). We performed several experiments to determine thenature of the modulation of [³⁵S]TBPS as affected by the compounds ofthe invention. We found that these compounds interact with a novel siteon the GR complex which does not overlap with the barbiturate, thebenzodiazepine or any other previously known sites. Furthermore, thesecompounds have high potency and efficacy at the GR complex, withstringent structural requirements for such activity. Preferred compoundswhich are useful in the present invention have an IC₅₀ of 2 μM or lessin the [³⁵S]TBPS binding assay described herein below.

The procedures for performing this assay are fully discussed in: (1)Gee, et al., 1987; and (2) Gee, K. W., L. J. Lawrence, and H. I.Yamamura, “Modulation of the chloride ionophore by benzodiazepinereceptor ligands: influence of gamma-aminobutyric acid and ligandefficacy,” Molecular Pharmacology 30:218, 1986. These procedures wereperformed as follows:

Brains from male Sprague-Dawley rats were removed immediately followingsacrifice and the cerebral cortices dissected over ice. A P₂ homogenatewas prepared as previously described (Gee, et al., 1986). Briefly, thecortices were gently homogenized in 0.32 M sucrose followed bycentrifugation at 1000×g for 10 minutes. The supernatant was collectedand centrifuged at 9000×g for 20 minutes. The resultant P₂ pellet wassuspended as a 10% (original wet weight/volume) suspension in 50 mM Na/Kphosphate buffer (pH 7.4) 200 mM NaCl to form the homogenate.

One hundred microliter (ml) aliquots of the P₂ homogenate (0.5milligrams (mg) protein) were incubated with 2 nanomolar (nM) [³⁵S]TBPS(70-110 curies/millimole;, New England Nuclear, Boston, Mass.) in thepresence or absence of the naturally occurring steroids or theirsynthetic derivatives to be tested. The tested compounds were dissolvedin dimethylsulfoxide (Baker Chem. Co., Phillipsbury, N.J.) and added tothe incubation mixture in 5 μL aliquots. The incubation mixture wasbrought to a final volume of 1 mL with buffer. Non-specific binding wasdefined as binding in the presence of 2 mM TBPS. The effect andspecificity of GABA (Sigma Chem. Co., St. Louis, Mo.) was evaluated byperforming all assays in the presence of GABA plus (+)bicuculline (SigmaChem. Co.). Incubations maintained at 25° C. for 90 minutes (steadystate conditions) were terminated by rapid filtration through glassfiber filters (No. 32, Schleicher and Schuell, Keene, NH). Filter-boundradioactivity was quantitated by liquid scintillation spectrophotometry.Kinetic data and compound/[³⁵S]TBPS dose-response curves were analyzedby nonlinear regression using a computerized iterative procedure toobtain rate constants and IC₅₀ (concentration of compound at whichhalf-maximal inhibition of basal [³⁵S]TBPS binding occurs) values.

The experimental data obtained for this assay are also published in Gee,et al., 1987. The data discussed in this reference are shown as plots inFIGS. 1A and 1B. These plots show the effect of (+)bicuculline on thepregnane steroid alphaxalone (1A) and GABA (1B) modulation of 2 nM[³⁵S]TBPS binding to rat cerebral cortex. In these figures, (◯)represents control without bicuculline; () represents 0.5 μMbicuculline; (□) represents 1.0 μM bicuculline; (▪) represents 2.0 μMbicuculline; and (Δ) represents 3.0 μM bicuculline. In this experiment,the effect of (+)bicuculline on the ability of alphaxalone or GABA toinhibit the binding of [³⁵S]TBPS was determined. Bicuculline is known tobe a competitive antagonist of GABA and a classical parallel shift inthe dose-response curves is observed in FIG. 1B. In contrast, thesteroid binding site identified by this work is distinct from theGABA/bicuculline site in FIG. 1A. The shift in dose-response curvesinduced by bicuculline when the inhibition of [³⁵S]TBPS binding iscaused by alphaxalone is not parallel. This indicates that the GABA andsteroid sites do not overlap.

A second set of experiments were performed to demonstrate that steroids,barbiturates and benzodiazepines do not share common binding site on theGABA receptor. FIG. 2 shows the time course for the dissociation of[³⁵S]TBPS from rat cortical P₂ homogenates initiated by the addition of2 μM TBPS(▪), 1 μM 3α-hydroxy-5α-pregnan-20-one (□), 100 μM Napentobarbital () and 1 μM 3α-OH-5α-pregnan-20-one +100 μM Napentobarbital (◯). The assay was performed in accordance with theprocedures outlined above. These kinetic data show that the dissociationof [³⁵S]TBPS binding initiated by saturating concentration of3α-hydroxy-5α-pregnan-20-one is potentiated by 100 μM Na pentobarbital.This effect is an indication that 3α-OH-5α-pregnan-20-one (steroid) andpentobarbital (barbiturate) bind to independent sites.

The third set of experiments examined the interactions between3α-hydroxy-5α-pregnan-20-one and Na pentobarbital in the potentiation of(³H) flunitrazepam (FLU) binding. These experiments further support theclaim that steroids do not share common site of action withbenzodiazepines and barbiturates. In this series of experiments, theeffect of varying concentrations of 3α-hydroxy-5α-pregnan-20-one on (³H)FLU binding in the presence or absence of a maximally stimulatingconcentration of Na pentobarbital. Since Na pentobarbital has greatermaximum efficacy than that of 3α-hydroxy-5α-pregnan-20-one inpotentiating (³H)FLU binding, 3α-hydroxy-5α-pregnan-20-one shouldultimately antagonize the effect of Na pentobarbital if the two interactcompetitively on the same site. This is not what was observed (FIG. 3).Thus, the data further support our conclusion that certain steroidsincluding the compounds of and used in the invention interact with anovel site distinct from the barbiturate or BZ regulatory site on the GRcomplex. Because of this independent site of action, it is anticipatedthat these steroid compounds will have therapeutic profiles differentfrom those of barbiturates and BZs.

Various compounds were screened to determine their potential asmodulators of [³⁵S]TBPS binding in vitro. These assays were performed inaccordance with the above discussed procedures. Based on these assays,we have established the structure-activity requirements for theirspecific interaction at the GR complex and their rank order potency andefficacy. FIG. 4 provides [³⁵S]TBPS inhibition of curves of3α-hydroxy-5α-pregnan-20-one (3α,5α-P),3α,21-dihydroxy-5α-pregnan-20-one (5α-THDOC) and R5020 (promogesterone)as experimental examples, while Table 1 provides IC₅₀ and maximuminhibition of numerous compounds, including examples of those claimed inthe application. IC₅₀ is defined as concentration of compounds toinhibit 50% of control [³⁵S]TBPS binding. It is an indication of acompound's in vitro potency. Maximum inhibition is an indication of acompound's in vitro efficacy.

TABLE 1 IC₅₀ IMAX Compound (nM) (%)3β-(4′-Acetylphenylethynyl)-3α,21-dihydroxy-5α-pregnan-20- 4 92 one3β-(4′-Acetylphenyl)ethynyl-3α-hydroxy-5α-pregnan-20-one 5 933β-(4′-Acetylphenyl)ethynyl-3α-hydroxy-5β-19-norpregnan- 6 90 20-one3β-(4′-Carboxyphenyl)ethynyl-3α-hydroxy-5α-pregnan-20- 7 79 one ethylester 3β-(4′-Carboxyphenyl)ethynyl-3α-hydroxy-5β-pregnan-20- 8 98 oneethyl ester 3β-(4-Hydroxybutyn-1-yl)-3α-hydroxy-5β-pregnan-20-one 13 1033β-(4′-Acetylphenyl)ethynyl-3α-hydroxy-5β-pregnan-20-one 14 1033β-(4′-Carboxyphenyl)ethynyl-3α-hydroxy-19-nor-5β- 15 94 pregnan-20-oneethyl ester 3β-(4-Acetoxybutyn-1-yl)-3α-hydroxy-5β-pregnan-20-one 20 963β-(4′-Acetylphenylethynyl)-3α,21-dihydroxy-5β-pregnan-20- 22 99 one3β-(4-Hydroxybutyn-1-yl)-3α-hydroxy-5β-19-norpregnan-20- 22 102 one3β-(3′-Methoxy-1′-propynyl)-3α-hydroxy-5β-pregnan-20-one 27 1053β-(4′-Dimethylaminophenyl)ethynyl-5β-pregnan-20-one 28 773β-[3-(2-propynyloxy)propyn-1-yl]-3α-hydroxy-5β-pregnan- 28 105 20-one3β-(4-Hydroxybutyn-1-yl)-3α-hydroxy-5α-pregnan-20-one 35 953α-Hydroxy-5α-pregnan-20-one 37 953β-(4′-Biphenyl)ethynyl-3α-hydroxy-5β-pregnan-20-one 43 833α-Hydroxy-3β-(4′-nitrophenyl)ethynyl-5β-pregnan-20-one 46 1033α-Hydroxy-21-(2H-1,2,3,4-tetrazol-2-yl)-5β-pregnan-20-one 46 783α-Hydroxy-3β-(4′-methoxyphenyl)ethynyl-5β-pregnan-20- 47 89 one3β-(4′-Trifluoromethylphenyl)ethynyl-3α-hydroxy-5β- 52 87 pregnan-20-one3β-(5-Acetylthien-2-yl)ethynyl-3α-hydroxy-5β-pregnan-20- 52 89 one21-(1-Benzimidazolyl)-3α-hydroxy-5β-pregnan-20-one 53 1003β-(4-Hydroxybutyn-1-yl)-3α-hydroxy-21-(1-imidazolyl)-5β- 54 93pregnan-20-one 3α-Hydroxy-21-(2H-1,2,3-triazol-2-yl)-5,6-pregnan-20-one56 79 3β-(4′-Hydroxybutyn-1′-yl)-3α-hydroxy-21-(1,2,3-triazol-2- 56 93yl)-5β-19-norpregnan-20-one3β-(4′-Chlorophenyl)ethynyl-3α-hydroxy-5β-pregnan-20-one 58 893β-(5′-Hydroxypentyn-1′-yl)-3α-hydroxy-5β-pregnan-20-one 58 1003α-Hydroxy-3β-(thien-2-yl)ethynyl-5β-pregnan-20-one 59 1003β-(3′-Acetylphenyl)ethynyl-3α-hydroxy-5β-pregnan-20-one 62 1003α-Hydroxy-21-[1H-(4-methyl-5-carboxyl)imidazol-1-yl)]-5β- 62 100pregnan-20-one 3α-Hydroxy-3β-[3′-(2H-1,2,3-triazol-2-yl)-1-propynyl]-5β-66 98 pregnan-20-one3α-Hydroxy-21-(9H-1,2,3-purin-9-yl)-5β-pregnan-20-one 69 593α-Hydroxy-3β-[4′(R/S)-hydroxypentynyl]-5β-pregnan-20- 71 103 one3α-Hydroxy-21-(imidazol-1-yl)-5α-pregnan-20-one 71 993α-Hydroxy-21-(imidazol-1-yl)-5β-pregnan-20-one 73 573β-(4′-Cyanophenyl)ethynyl-3α-hydroxy-5β-pregnan-20-one 73 923α,21-Dihydroxy-5α-pregnan-20-one (5α-THDOC) 76 1003α-Hydroxy-3β-(pentafluorophenyl)-ethynyl-5β-pregnan-20- 79 69 one3β-(4′-Hydroxybutyn-1′-yl)-3α-hydroxy-21-(1,2,3-triazol-1- 79 96yl)-5β-pregnan-20-one3β-Ethynyl-3α-hydroxy-21-(1′-imidazolyl)-5β-pregnan-20-one 80 1033α-Hydroxy-3β-[3′-(1H-pyrazol-1-yl)-1′-propynyl]-5β- 81 98pregnan-20-one 3α-Hydroxy-3β-methyl-21-(2H-1,2,3-triazol-2-yl)-5α- 85 99pregnan-20-one 3β-[4′(N,N-Diethylcarboxamidophenyl)ethynyl]-3α-hydroxy-87 94 5β-pregnan-20-one3α-Hydroxy-3β-(4′-methylphenyl)ethynyl-5β-pregnan-20-one 88 843α-Hydroxy-3β-(6-oxo-1-heptynyl)-5β-pregnan-20-one 90 1013β-(4′-Hydroxybutyn-1′-yl)-3α-hydroxy-21-(1,2,4-triazol-1- 93 96yl)-5β-pregnan-20-one3β-(4′-Hydroxybutyn-1′-yl)-3α-hydroxy-21-(tetrazol-1-yl)-5β- 93 101pregnan-20-one 3α-Hydroxy-3β-[4′(R/S)-hydroxypentynyl]-5β-pregnan-20- 94104 one 3β-(5′-Acetoxypentyn-1′-yl)-3α-hydroxy-5β-pregnan-20-one 96 983α-Hydroxy-21-(1H-3,5-dimethylpyrazolyl)-5β-pregnan-20- 96 77 one3α-Hydroxy-21-(1′-imidazolyl)-3β-methyl-5α-pregnan-20-one 97 953α-Hydroxy-3β-(phenylethynyl)-5β-pregnan-20-one 98 1013β-(4′-Hydroxybutyn-1′-yl)-3α-hydroxy-21-(1′-imidazolyl)- 98 985β-19-norpregnan-20-one 3α-Hydroxy-21(1′-pyrazolyl)-5α-pregnan-20-one100 98 3β-(2′-Hydroxyphenyl)ethynyl-3α-hydroxy-5β-pregnan-20- 101 83 one3α-Hydroxy-3β-(3′-phenyl-1′-propynyl)-5β-pregnan-20-one 103 953β-Ethynyl-3α-Hydroxy-21-(1′-pyrazolyl)-5β-pregnan-20-one 106 99 SodiumS-[3α-hydroxy-3β-(4′-hydroxybutynyl)-5β-pregnan- 107 97 20-on-21-yl]thiosulfate 3α-Hydroxy-3β-(2′-pyridyl)ethynyl-5β-pregnan-20-one 108 983β-(2,4-Difluorophenyl)ethynyl-3α-hydroxy-5β-pregnan-20- 109 102 one3β-Benzyl-3α-hydroxy-5β-pregnan-20-one 109 102 20-one21-(1′-Benzimidazolyl)-3α-hydroxy-3β-methyl-5α-pregnan- 109 95 20-one3α-Hydroxy-21-(1′-pyrazolyl)-3β-methyl-5α-pregnan-20-one 115 983α-Hydroxy-3β-5′-oxo-1-hexynyl)-5β-pregnan-20-one 118 1013α-Hydroxy-21-(pyrazol-1-yl)-5β-pregnan-20-one 121 673β-(4′-Hydroxybutyn-1′-yl)-3α-hydroxy-21-(1,2,3-triazol-1- 124 95yl)-5β-19-norpregnan-20-one3α-Hydroxy-3β-methyl-21-(1′,2′,4′-triazolyl)-5α-pregnan-20- 125 105 one3β-Ethynyl-3β-hydroxy-21-(1′,2′,4′-triazolyl)-5β-pregnan-20- 127 98 one3β-(4′-Cyanobutynl)-3α-hydroxy-5β-pregnan-20-one 131 86 SodiumS-[3α-hydroxy-3β-methoxymethyl)-5β-pregnan-20- 132 103 on-21-yl]thiosulfate Sodium S-(3α-hydroxy-5α-pregnan-20-n-21-yl)thiosulfate 13390 3α-Hydroxy-3β-(2′-phenylethyl)-5β-pregnan-20-one 135 983α-Hydroxy-3β-methyl-21-(1′,2′,3′-triazol-1′-yl)-5α-pregnan- 136 9920-one 3α-Hydroxy-3β-[3′-hydroxypropynyl]-5β-pregnan-20-one 137 933α-Hydroxy-3β-(3′-(3-pyridyl)ethynyl-5β-pregnan-20-one 138 1003α-Hydroxy-3β-[3′(RS)-hydroxybutynyl]-5α-pregnan-20-one 144 993α-Hydroxy-3β-(4-pyridyl)ethynyl-5β-pregnan-20-one 149 1033α-Hydroxy-21-(1′,2′,4′-triazol-1-yl)-5β-pregnan-20-one 151 60 SodiumS-(3α-hydroxy-3β-methyl-5α-pregnan-20-on-21- 151 101 yl)thiosulfate3β-(5′-Cyanopentynyl)-3α-hydroxy-5β-pregnan-20-one 158 1003β-(4′-Acetoxyacetylphenyl)ethynyl-3α-hydroxy-5β-pregnan 171 91 20-one3α-Hydroxy-3β-phenylpropyl-5β-pregnan-20-one 173 923α-Hydroxy-3β-[4′(R/S)-hydroxypentynyl]-5α-pregnan-20- 178 101 one3β-hydroxy-3β-[3′(RS)-hydroxybutynyl]-5β-pregnan-20-one 202 1023β-(6-Hydroxyhexyn-1-yl)-3α-hydroxy-5β-pregnan-20-one 222 993α-Hydroxy-21-[1H-2-methyl)imidazol-1-yl)-5β-pregnan-20- 222 95 one3β-(3′-Acetoxypropyn-1′-yl)-3α-hydroxy-5β-pregnan-20-one 224 1043α-hydroxy-21-(1′H-1,2,3,4-tetrazol-1′-yl)-5β-pregnan-20- 227 60 one3α-Hydroxy-21-(2-formylimidazol-1-yl)-5β-pregnan-20-one 213 813α-Hydroxy-3β-(2′-methoxyphenyl)ethynyl-5β-pregnan-20- 238 99 one3β-(4′-hydroxy-1′-butynyl)-3α-hydroxy-5β-pregnan-11,20- 268 102 dione3β-[(3′,4′-Dimethoxyphenyl)ethynyl)-3α-hydroxy-5β- 283 106pregnan-20-one 3α-Hydroxy-21-[1H-4-nitro)imidazol-1-yl)]-5β-pregnan-20-267 71 one Sodium S-(3α-hydroxy-5β-pregnan-20-on-21-yl)thiosulfate 26888 3β-(6′-Acetoxyhexyn-1′-yl)-3α-hydroxy-5β-pregnan-20-one 306 99 SodiumS-(3α-hydroxy-3β-trifluoromethyl-5β-19-norpregnan- 325 98 20-on-21-yl)thiosulfate 3α-Hydroxy-3β-phenyl-5β-pregnan-20-one 382 863β-(3′-Hydroxyphenyl)ethynyl-3α-hydroxy-5β-pregnan-20- 407 99 one3α-Hydroxy-3β-(3-hydroxypropyn-1-yl)-21-(1,2,3-triazol-2- 430 107yl)-5β-pregnan-20-one 3α-Hydroxy-21-(7′H-purin-7′-yl)-5β-pregnan-20-one430 71 3α-Hydroxy-21-(1′-uracil)-5α-pregnan-20-one 434 903α-Hydroxy-3β-[3′-(pyrid-4-yloxy)-1′-propynyl]-5β-pregnan- 465 94 20-one3β-[2-(3′,4′-Dimethoxyphenyl)ethyl]-3α-hydroxy-5β-pregnan- 507 66 20-one3α-Hydroxy-3β-(3′-hydroxyphenyl)-ethynyl-5β-pregnan-20- 619 98 one3α-Hydroxy-3β-[3′-(1H-1,2,3-triazol-1-yl)-1′-propynyl]-5β- 655 102pregnan-20-one3α-Hydroxy-3β-[3′-(1H-1,2,4-triazol-1-yl)-1′-propynyl]-5β- 843 99pregnan-20-one 3α-Hydroxy-3β-(3-hydroxypropyn-1-yl)-21-(1,2,3-triazol-1-1089 101 yl)-5β-pregnan-20-one3β-[3′-(N,N-dimethylamino)-1′-propynyl]-3β-hydroxy-5β- 1420 100pregnan-20-one 3β-(4′-Acetoxyphenyl)ethynyl-3α-hydroxy-5β-pregnan-20-one1430 100 3β-(4-Hydroxyphenyl)ethynyl-3α-hydroxy-5β-pregnan-20-one 155088 3β-Hydroxy-2β-morpholinyl-21-(1H-1,2,4-triazol-1-yl)-5α- 1553 97pregnan-20-one 3β-Hydroxy-3β-(3′-oxopropynyl)-5β-pregnan-20-one 1720 763β-(4′-Carboxamidophenyl)ethynyl-3α-hydroxy-5β-pregnan- 2240 98 20-one3β-(4′-Carboxamidobutynyl)-3α-hydroxy-5β-pregnan-20-one 2340 1023α-Hydroxy-3β-(3′-oxobutynyl)-5β-pregnan-20-one 2690 1073α-Hydroxy-3β-[3′-(N-imidazolyl)-1′-propynyl]-5β-pregnan- 2720 10720-one 3β-(5′-Carboxamidopentynyl)-3α-hydroxy-5β-pregnan-20-one 3090 102Progesterone 5200 100 3β-Hydroxy-3β-[3′-oxobutynyl]-5α-pregnan-20-one6510 104 3β-(4′-Aminophenyl)ethynyl-3β-hydroxy-5β-pregnan-20-one 22500103 3β-Hydroxy-5α-pregnan-20-one (Allopregnanolone) >10⁶ 334-Pregnen-11β,21-diol-3,20-dione (Corticosterone) >10⁶ 30 17β-Estradiolna^(a) 0 Cholesterol na 0 ^(a)na = not active

As can be seen from FIG. 4 and Table 1, 3α-hydroxy-5α-pregnan-20-one,3α,21-dihydroxy-5α-pregnan-20-one and compounds of and used in theinvention have low IC₅₀, which is the concentration necessary to achieve50% maximal inhibition of [³⁵S]TBPS binding, while compounds such as sexsteroids (R5020, estradiol and progesterone), glucocorticoids(corticosterone) and cholesterol having a high IC₅₀ are essentiallyinactive. Thus, it is anticipated that hormonal steroids and cholesterolper se will not have any therapeutic value for the indications describedherein. In order to distinguish this unique class of steroids fromhormonal steroids, they are now termed neuroactive steroids. However,sex steroids such as progesterone can be metabolized in the body tosteroids similar to 3α-hydroxy-5α-pregnan-20-one. Thus, progesterone canbe considered as a prodrug. The TBPS data correlates with date on ³⁶Clion uptake-potentiated by various 3α-hydroxylated steroids described inPurdy R. H., et al., “Synthesis, Metabolism, and PharmacologicalActivity of 3α-Hydroxy Steroids Which Potentiate GABA-Receptor-MediatedChloride Ion Uptake in Rat Cerebral Cortical Synaptoneurosomes,” J. Med.Chem 33:1572-1581 (1990), incorporated herein by reference and thesedata also correlate with electrophysiological data obtained by measuringsteroid's activity to potentiate GABA-induced current in oocytesinjected with human GABA receptors as shown in FIG. 5. This indicatesthat the TBPS assay is an approximate measurement of steroids ability toallosterically modulate Cl⁻ channel activity.

Compounds with Limited Efficacy

In as much as the desired therapeutic activity should be available tothe patient with the least undesirable side effects, a notable aspect ofthis invention involves the discovery of agonists with partial activityin those compounds with a 5α-pregnan-3α,20α-diol, 5β-pregnan-3α,20β-diolgroup or the derivatives and prodrugs of these compounds. In addition, asubset of neuroactive steroids other than these two groups also showpartial efficacy in TBPS assay (Table 1). For the patients who desireamelioration of anxiety or convulsions, hypnosis is undesired. For thepatients who desire amelioration of insomnia, anesthetic is undesired.The compounds and activities described as agonists with partial activityexpected to have the desired effect with minimal undesired effect.

To show the agonists with limited efficacy, the ability of5α-pregnan-3α,20α-diol and 5β-pregnan-3α,20α-diol to partially modulatethe [³⁵S]TBPS binding even at very high concentrations is illustrated(FIG. 6)

In addition, the partial ability of these compounds to potentiateGABA-mediated enhancement of Cl⁻ current were compared to that of3α-hydroxy-5α-pregnan-20-one (FIG. 7) in Xenopus oocyte injected withhuman GABA_(A) receptor genes is also shown.

When Xenopus oocyte expression system was used to test the limitefficacy property of some neuroactive steroids, the following procedurewas performed. Xenopus laevis oocytes (stage VI) which had been“defolliculated” using the collagenase digestion method (3 hrs @ 18-23°C., 2 mg ml⁻¹ collagenase ‘A’ in Barth's saline with Ca²⁺ salts omitted)were injected with cRNA transcripts of human GABA_(A) receptor subunitcomplex α1 β1 and γ1. The major GABA_(A) receptor complex is comprisedof αβγ subunits. Injected oocytes were individually maintained in96-well plates (200 μL per well of normal Barth's solution supplementedwith penicillin 50 IU ml⁻¹, streptomycin 50 mg ml⁻¹ and gentomycin 100mg ml⁻¹) for up to 9 days at 19-20° C. Agonist-induced currents wererecorded from Xenopus oocytes voltage clamped at a holding potential of−60 mV, using an Axoclamp 2 A (Axon Instruments) voltage clamp amplifierin the twin electrode voltage clamp mode. The voltage-sensing andcurrent-passing microelectrodes were filled with 3M KCl, and resistancesof 1-3 M Ohams when measured in the standard extracellular saline. Theoocytes were continuously superfused with frog Ringer (120 mM NaCl; 2 mMKCl; 1.0 mM CaCl₂; 5 mM HEPES pH 7.4) at the rate of 5-7 ml min⁻¹ at rt(17-21° C.).

All drugs were applied via the perfusion system. Steroids (10⁻² M) wereprepared as concentrated stock solutions either in DMSO or ethanol andthen diluted in the Ringer solution at the appropriated concentration.The final DMSO and ethanol concentration was 0.2% v/v, a concentrationwhich had no effect upon GABA evoked responses. Stock solutions of allother drugs were made in Ringer solution. Membrane current responseswere low-pass filtered at 100 Hz and recorded onto magnetic tape usingan FM tape recorder (Racal Store 4DS) for subsequent analysis.

Compounds of and used in the present invention exhibit partial efficacyanalogous to those described above also shown in the above tables.

Benefits Over Progesterone

The correlations between reduced levels of progesterone and the symptomsassociated with PMS, PND, and catamenial epilepsy (Backstrom, et al.,1983; Dalton, K., 1984) led to the use of progesterone in theirtreatment (Mattson, et al., 1984; and Dalton, 1984). However,progesterone is not consistently effective in the treatment of theaforementioned syndromes. For example, no dose-response relationshipexists for progesterone in the treatment of PMS (Maddocks, et al.,1987). These results are predictable when considered in light of theresults of our in vitro studies which demonstrate that progesterone hasvery low potency at the GR complex, as seen in Table 1, compared tocertain metabolites of progesterone.

The beneficial effect of progesterone is probably related to thevariable conversion of progesterone to the active progesteronemetabolites. The use of specific progesterone metabolites in thetreatment of the aforementioned syndromes is clearly superior to the useof progesterone based upon the high potency and efficacy of themetabolites and their derivatives (See Gee, et al., 1987, and Table 1).

No Hormonal Side Effects

It has also been demonstrated that neuroactive steroids lack hormonalside effects by the lack of affinity for the progesterone and otherhormonal steroid receptors (Tables 2-5). The data presented wereobtained by performing assays in accordance with the proceduresdescribed in Gee, et al. 1988 previously to determine the effect ofprogesterone metabolites and their derivatives and the progestin R5020on the binding of [³H]R5020 to the progesterone receptor in rat uterus(Gee et al. 1988).

³H-progesterone (0.15 nM) was incubated with the rat uterus cytosol inthe presence of the test compounds. The specific bindings weredetermined after incubation and compared to the control incubationwithout the compounds. The data are expressed as percent inhibition ofbinding. If the compounds bind to the progesterone receptor with highaffinity, a 100% inhibition of binding would be expected at theconcentration tested.

Various hormonal activities of representative neuroactive steroids werefurther studied through testing their potential estrogenic,mineralocorticoid and glucocorticoid activities. These activities wereanalyzed by monitoring the ability of the compounds to inhibit bindingof the steroid hormones to their respective hormone receptors. Theresults are shown in Tables 3-5. They are expressed as percentinhibition of ³H-ligand binding to the various steroid hormone receptorsfor the compounds at 10⁻⁶ M. Control values are represented by thebinding in the absence of testing compounds.

In Table 3, rats were adrenalectomized 3 days prior to sacrifice. Toisolate the mineralocorticoid receptor, brain cytosol fractions wereprepared as describe in Gee, et al. 1988. The drugs were incubated with3 nM of ³H-aldosterone (the specific ligand for the mineralocorticoidreceptor) in the presence of the selective type II agonist RU28362 (0.5μM) which blocks ³H-aldosterone binding to the type II (glucocorticoid)receptors.

TABLE 2 Inhibition of ³H-Progesterone Binding to the Bovine UteralProgesterone Receptors Competitor (10⁻⁶M) % of Inhibition R5020 100 5α-pregnan-3α-ol-20-one 14  5α-pregnan-3α,21-diol-20-one 13 5α-pregnan-3α,20-diol 6 5α-pregnan-3α-ol-3α,-methyl-20-one 45β-pregnan-3α,21-diol-20-one 6 5α-pregnan-3β,20-trimethyl-3α,20-diol 85β-pregnan-3α,20α-diol 0 5β-pregnan-3α-ol-20-one 95α-pregnan-20-dimethyl-3α,20-diol 0

TABLE 2 Inhibition of ³H-Progesterone Binding to the Bovine UteralProgesterone Receptors Competitor (10⁻⁶M) % of Inhibition R5020 100 5α-pregnan-3α-ol-20-one 14  5α-pregnan-3α,21-diol-20-one 13 5α-pregnan-3α,20-diol 6 5α-pregnan-3α-ol-3α,-methyl-20-one 45β-pregnan-3α,21-diol-20-one 6 5α-pregnan-3β,20-trimethyl-3α,20-diol 85β-pregnan-3α,20α-diol 0 5β-pregnan-3α-ol-20-one 95α-pregnan-20-dimethyl-3α,20-diol 0

For Table 4, brain cytosol fractions were prepared as for Table 3, andthe compounds were incubated with 3 nM of ³H-dexamethasone (the specificligand for the glucocorticoid receptor).

TABLE 4 Inhibition of ³H-Dexamethasone Binding to GlucocorticoidReceptors Competitor (10⁻⁶M) % or Inhibition Dexamethasone 1005α-pregnan-3α,21-diol-20-one 29.5 5β-pregnan-3α,21-diol-20-one 8.25α-pregnan-3α-ol-20-one 8.7 5β-pregnan-3α-ol-20-one 5.95α-pregnan-3α,20α-diol 2.6 5β-pregnan-3α,20α-diol 1.45α-pregnan-20-dimethyl-3α,20-diol 2.6 5α-pregnan-3α-ol-3β-methyl-20-one0.6

Table 5 shows the inhibition of ³H-estradiol (the specific ligand forthe estrogen receptor) binding to bovine uteri cytosol, prepared aspreviously described (Gee, et al. 1988). ³H-Estradiol (0.15 nM) wasincubated with the cytosol in the presence of the compounds.

TABLE 5 Inhibition of ³H-Estradiol Binding to Bovine Uteral EstrogenReceptors Competitor (10⁻⁶M) % of Inhibition 17β-estradiol 100 5α-pregnan-3α-ol-20-one 0 5α-pregnan-3α,21-diol-20-one 25α-pregnan-3α,20α-diol 0 5α-pregnan-3α-ol-3-methyl-20-one 05β-pregnan-3α,21-diol-20-one 0 5α-pregnan-3β,20-trimethyl-3α,20-diol 05β-pregnan-3α,20α-diol 8 5β-pregnan-3α-ol-20-one 05α-pregnan-20-dimetfhyl-3α,20-diol 0

The results of these experiments clearly show that neuroactive steroidsdo not have a strong affinity for any of the above steroid receptors.Thus, they will not have predicted hormonal side-effects which wouldresult from such steroid receptor binding.

Anti-Convulsant Activity

Experiments were also performed to determine the physiological relevanceof neuroactive steroid and GABA receptor interactions by assessing theability of the compounds of and used in the invention to preventmetrazol induced convulsions in mice. Mice were injected with variousdoses of the test compounds of the invention, 10 minutes prior to theinjection of metrazol. The time to onset of myoclonus (presence offorelimb clonic activity) induced by metrazol was determined byobserving each mouse for a period of 30 minutes. In control mice,metrazol (85 mg/kg) will induce convulsion in 95% of the animals. Theability of several compounds of and used in the invention to protectmice from convulsion is shown in Table 6.

TABLE 6 Antimetrazol Activity of Neuroactive Steroids in Mice Dose %Name Route Vehicle (mg/kg) Protected3β-(4′-Acetylphenyl)ethynyl-3α-hydroxy-5α- IP 50% 10 75 pregnan-20-onehpbcd 3β-(4′-Acetylphenyl)ethynyl-3α-hydroxy-19-nor-5β- IP 50% 10 100pregnan-20-one hpbcd 3β-(4′-Carhoxyphenyl)ethynyl-3α-hydroxy-5β- IP 50%10 18.75 pregnan-20-one ethyl ester hpbcd3β-(4′-Hydroxybutyn-1′-yl)-3α-hydroxy-5β-pregnan- IP 50% 10 100 20-onehpbcd 3β-(4′-Acetylphenyl)ethynyl-3α-hydroxy-5β- IP 50% 10 75pregnan-20-one hpbcd 3β-(4′-Acetoxybutyn-1′-yl)-3α-hydroxy-5β-pregnan-IP 50% 10 100 20-one hpbcd 3α-Hydroxy-3β-(3′-methoxy-1′-propynyl)-5β- IP50% 10 75 pregnan-20-one hpbcd3β-(4′-Acetylphenylethynyl)-3α,21-dihydroxy-5α- IP 50%  1 87.5pregnan-20-one hpbcd 3β-(4′-Acetylphenylethynyl)-3α,21-dihydroxy-5β- IP50% 10 75 pregnan-20-one hpbcd3β-(4′-Acetylphenylethynyl)-3α,21-dihydroxy-5α- PO water 10 80pregnan-20-one 21-hemisuccinate salt3β-(4′-Acetylphenylethynyl)-3α,21-dihydroxy-5β- PO water 10 75pregnan-20-one 21-hemisuccinate salt3β-(4′-hydroxy-1′-butynyl)-3α-hydroxy-5β-pregnan- IP 50% 10 5011,20-dione hpbcd 3β-[3-(2-propynyloxy)propyn-1-yl]-3α-hydroxy-5β- IP50% 10 50 pregnan-20-one hpbcd3β-(4′-Hydroxybutyn-1′-yl)-3α-hydroxy-5α-pregnan- IP 50% 10 50 20-onehpbcd 3α-Hydroxy-3β-(5′-oxo-1-hexynyl)-5β-pregnan-20- IP 50% 10 50 onecyclic 5′-(1,2-ethanediyl acetal) hpbcd3β-(4′-Biphenyl)ethynyl-3α-hydroxy-5β-pregnan-20- IP dmso 10 0 one3β-(5-Acetylthien-2-yl)ethynyl-3α-Hydroxy-5β- IP 50% 10 25pregnan-20-one hpbcd 3β-(4′-Trifluoromethylphenyl)ethynyl-3α-hydroxy- IPdmso 10 25 5β-pregnan-20-one 3β-(4′-Chlorophenyl)ethynyl-3α-hydroxy-5β-IP dmso 10 37.5 pregnan-20-one3β-(5′-Hydroxypentyn-1′-yl)-3α-hydroxy-5β- IP 50% 10 12.5 pregnan-20-onehpbcd 3α-Hydroxy-3β-(thien-2-yl)ethynyl-5β-pregnan-20- IP 50% 10 37.5one hpbcd 3β-(3′-Acetylphenyl)ethynyl-3α-hydroxy-5β- IP 50% 10 75pregnan-20-one hpbcd 3α-Hydroxy-3β-[4′(R′S)-hydroxypentynyl]-5β- IP 50%10 87.5 pregnan-20-one hpbcd 3β-(4′-Cyanophenyl)ethynyl-3α-hydroxy-5β-IP 50% 10 43.7 pregnan-20-one hpbcd3β-(4′-Hydroxybutyn-1′-yl)-3α-hydroxy-5β-pregnan- IP water 10 87.520-one 4′-hemisuccinate sodium salt3α-Hydroxy-3β-[3′-(1H-pyrazol-1-yl)-1′-propynyl]- IP 50% 10 255β-pregnan-20-one hpbcd 3α-Hydroxy-3β-(4′-methylphenyl)ethynyl-5β- IP50% 10 75 pregnan-20-one hpbcd3α-Hydroxy-3β-(6-oxo-1-heptynyl)-5β-pregnan-20- IP 50% 10 62.5 one hpbcd3β-(5′-Acetoxypentyn-1′-yl)-3α-hydroxy-5β- IP 50% 10 12.5 pregnan-20-onehpbcd 3α-Hydroxy-3β-(2′-pyridylethynyl)-5β-pregnan-20- IP 50% 10 37.5one hpbcd 3α-Hydroxy-3β-(5′-oxo-1-hexynyl)-5β-pregnan-20- IP 50% 10 25one hpbcd 3β-(4′-Cyano-1′-butynyl)-3α-hydroxy-5β-pregnan- IP 50% 10 12.520-one hpbcd 3α-Hydroxy-3β-(3′-hydroxypropynyl)-5β-pregnan- IP 50% 10 2520-one hpbcd 3α-Hydroxy-3β-[3′(RS)-hydroxybutynyl]-5α- IP 50% 10 50pregnan-20-one hpbcd 3α-Hydroxy-3β-[4′(R/S)-hydroxypentynyl]-5β- IPwater 10 50 pregnan-20-one 4′(R/S)-hemisuccinate sodium salt3β-(5′-Cyanopentynyl)-3α-hydroxy-5β-pregnan-20- IP 50% 10 25 one hpbcd3α-Hydroxy-3β-[4′(R/S)-hydroxypentynyl]-5α- IP 50% 10 25 pregnan-20-onehpbcd 3α-Hydroxy-3β-[3′(RS)-hydroxybutynyl]-5β- IP 50% 10 25pregnan-20-one hpbcd 3β-(5′-Hydroxypentyn-1′-yl)-3α-hydroxy-5β- IP 50%10 37.5 pregnan-20-one 5′-hemisuccinate sodium salt hpbcd3β-(3′-Acetoxypropyn-1′-yl)-3α-hydroxy-5β IP 50% 10 12.5 pregnan-20-onehpbcd 3α-Hydroxy-3β-(2′-methoxyphenyl)ethynyl-5β- IP dmso 10 12.5pregnan-20-one 3β-(6′-Acetoxyhexyn-1′-yl)-3α-hydroxy-5(3-pregnan- IP 50%10 37.5 20-one hpbcd 3α-Hydroxy-3β-[3′-(pyrid-4-yloxy)-1′-propynyl]-5β-IP 50% 10 12.5 pregnan-20-one hpbcd3β-[2-(3′,4′-Dimethoxyphenyl)ethyl]-3α-hydroxy- IP 50% 10 12.55β-pregnan-20-one hpbcd 3β-(3′-Hydroxy-3′-methylbutyn-1′-yl)-3α-hydroxy-IP 50% 10 43.7 5β-pregnan-20-one hpbcd3α-Hydroxy-3β-[3′-(1H-1,2,3-triazol-1-yl)-1′- IP 50% 10 25propynyl]-5β-pregnan-20-one hpbcd3α-Hydroxy-3β-[3′-(1H-1,2,4-triazol-1-yl)-1′- IP 50% 10 10propynyl]-5β-pregnan-20-one hpbcd SodiumS-(3α-hydroxy-5β-pregnan-20-on-21-yl) IP water 10 37.5 thiosulfateSodium S-(3α-hydroxy-3β-methyl-5α-pregnan-20- IP water 10 62.5 on-21-yl)thiosulfate Sodium S-[3α-hydroxy-3β-(4′-hydroxybutynyl)-5β- PO water 4033.3 pregnan-20-on-21-yl] thiosulfate SodiumS-(3α-hydroxy-3β-methoxymethyl-5α- IP water 10 62.5 pregnan-20-on-21-yl)thiosulfate Sodium S-(3α-hydroxy-3β-trifluoromethyl-19-nor- IP water 4037.5 5β-pregnan-20-n-21-yl)thiosulfate3α-Hydroxy-21-(1′-imidazolyl)-5β-pregnan-20-one IP 50% 10 50hemisuccinate sodium salt hpbcd3α-hydroxy-21-(2′H-1,2,3,4-tetrazol-2′-yl)-5β- IP 50% 10 37.5pregnan-20-one hpbcd 3α-Hydroxy-21-(2H-1,2,3-triazol-2-yl)-5β-pregnan-IP 50% 10 100 20-one hpbcd3α-Hydroxy-21-[1H-(4-methyl-5-carboxyl)imidazol- IP 50% 10 37.51-yl)-5β-pregnan-20-one ethyl ester hpbcd21-[1′-(4,5-Dichloro)imidazolyl]-3α-hydroxy-5β- IP 50% 10 37.5pregnan-20-one hpbcd 3α-Hydroxy-21-(1′-imidazolyl)-5α-pregnan-20-one IP50% 10 50 hpbcd 3α-Hydroxy-21-(1′-imidazolyl)-5β-pregnan-20-one IP 50%10 12.5 hpbcd 3β-Ethynyl-3α-Hydroxy-21-(1′-imidazolyl)-5β- IP 50% 10 50pregnan-20-one hpbcd 3α-Hydroxy-21-(1H-3,5-diethylpyrazolyl)-5β- IP 50%10 50 pregnan-20-one hpbcd 3α-Hydroxy-21-(1′-imidazolyl)-3β-methyl-5α-IP 50% 10 75 pregnan-20-one hpbcd3,3-Ethynyl-3α-Hydroxy-21-(1′-pyrazolyl)-5β- IP 50% 10 62.5pregnan-20-one hpbcd 3α-Hydroxy-21-(1′-pyrazolyl)-3β-methyl-5α- IP 50%10 87.5 pregnan-20-one hpbcd3α-Hydroxy-22-(pyrazol-1-yl)-5β-pregnan-20-one IP 50% 10 62.5 hpbcd3α-Hydroxy-3β-methyl-21-(1′,2′,4′-triazolyl)-5α- IP 50% 10 87.5pregnan-20-one hpbcd 3α-Hydroxy-21-(1′,2′,4′-triazol-1-yl)-5β-pregnan-IP 50% 10 37.5 20-one hpbcd21-[1′-(4,5-Dicyano)imidazolyl]-3α-hydroxy-5β- IP 50% 10 50pregnan-20-one hpbcd 3α-Hydroxy-21-[1H-(2-methyl)imidazol-1-yl)-5β- IP50% 10 6.2 pregnan-20-one hpbcd3α-Hydroxy-21-(1′-pyrazolyl)-3β-trifluoromethyl- IP 50% 10 12.55β-19-nor-pregnan-20-one hpbcd3α-Hydroxy-21-(1′-imidazolyl)-3β-trifluoromethyl- IP 50% 10 505β-19-nor-pregnan-20-one hpbcd

The ability of neuroactive steroids to protect animals against otherchemical convulsants was further demonstrated for3α-hydroxy-5α-pregnan-20-one, 3α,21-dihydroxy-5α-pregnan-20-one and3α-hydroxy-3β-methyl-5α-pregnan-20-one. The anticonvulsant tests aresimilar to that described above. The following chemical convulsants are:metrazol (85 mg/kg); (+)bicuculline (2.7 mg/kg); picrotoxin (3.15mg/kg); strychnine (1.25 mg/kg); or vehicle (0.9% saline). Immediatelyafter the injection of convulsant or vehicle, the mice were observed fora period of 30 to 45 minutes. The number of animals with tonic and/orclonic convulsions was recorded. In the maximal electroshock test, 50 mAof current at 60 Hz was delivered through corneal electrodes for 200msec to induce tonic seizure. The ability of compounds to abolish thetonic component was defined as the end point. General CNS depressionpotential was determined by a rotorod test 10 minutes after theinjection of compounds where the number of mice staying on a rotating (6rpm) rod for 1 minute in one of the three trials was determined. TheED₅₀ (the dose at which the half-maximal effect occurs) was determinedfor each screen and are presented in Table 7, infra. The resultsdemonstrate that neuroactive steroids, in comparison to other clinicallyuseful anti-convulsants, are highly effective with profiles similar tothat of the BZ clonazepam. These observations demonstrate thetherapeutic utility of these compounds as modulators of brainexcitability, which is in correspondence with their high affinityinteraction with the GR complex in vitro.

TABLE 7 Anticonvulsant Activity of Exemplified Neuroactive Steroids andthose of Selected Clinically Useful Anticonvulsants in Mice ED₅₀ (mg/Kg)Compound RR MES MTZ BIC PICRO STR 3α5α^((a))-P 30 28.6 4.9 12.310.2 >300 5α-THDOC^((a)) 22.9 26.7 8.1 17.8 5.6 >300 3α-hydroxy-3β-246.2 >100 6.3 61.9 35.4 >100 methyl-5α-pregnan-20 one^((b)) Clonazepam*0.184 93 0.009 0.0086 0.043 NP Phenobarbital* 69 22 13 38 28  95Phenytoin* 65 10 NP NP NP ** Progabide*** — 75 30 30 105  75 Valproate*426 272 149 360 387  293 The abbreviations are RR (Rotorod); MES(maximal electroshock); MTZ (metrazol); BIC (bicuculline); PICRO(picrotoxin); STR (strychnine); NP (no protection) ^((a))Dissolved in20% hydroxypropyl-β-cyclodextrin in water. The route of administrationfor steroids and convulsants was i.p. and s.c., respectively.*Anticonvulsant data are from Swinyard & Woodhead, General principles:experimental detection, quantification and evaluation ofanticonvulsants, in Antiepileptic Drugs, D. M. Woodbury, J. K. Penry,and C. E. Pippenger, eds. p. 111, (Raven Press, New York), 1982.^((b))Vehicle contained 0.32% hydroxypropylmethyl cellulose and 4% tween80 in saline. **Maximum protection of 50% at 55-100 mg/kg. ***Thechemical convulsants in the progabide studies were administered i.v.,all data from Worms et al., Gamma-amninobutyric acid (GABA) receptorstimulation. I. Neuropharmacological profiles of progabide (SL 76002)and SL 75102, with emphasis on their anticonvulsant spectra, Journal ofPharmacology and Experimental Therapeutics 220: 660-671 (1982).

Anxiolytic Effects

The following experiments demonstrate that the progesterone metabolites,3α-OH-5α-pregnan-20-one and 3α-OH-5β-pregnan-20-one are effectiveanxiolytics in four animal models of human anxiety that measure thebehavioral effects of anxiolytic compounds. It is to be understood thatthese two compounds describe the invention by way of illustration. Dataon their synthetic derivatives in these measurements also is presentedin Tables 8-10. The four animal models used to measure the behavioraleffects of anxiolytic compounds are: 1) light/dark transition test; 2)elevated plus-maze; 3) Geller-Seifter conflict test and 4) Vogel test.

a) Light/dark Transition Test

The light/dark transition test (Crawley and Goodwin, “Preliminary reportof a simple animal behavior model for the anxiolytic effect ofbenzodiazepines”, Pharmacol. Biochem. Behav. 13:67-70 (1980)) is basedon the observation that rodents naturally tend to explore novelenvironments, but open, brightly lit arenas are aversive to the rodentsand inhibit exploratory behavior (Christmas and Maxwell, “A comparisonof effects of some benzodiazepines and other drugs on aggressive andexploratory behaviour in mice and rats”, Neuropharmacol. 9:17-29 (1970);File, “The use of social interaction as a method of detecting anxiolyticactivity of chlordiazepoxide-like drugs”, J. Neurosci. Meth. 2:219-238(1980)). A variety of clinically established anxiolytics includingdiazepam, clonazepam and pentobarbital have been shown to increase thenumber of transitions between the light box and the dark box, whereasnon-anxiolytic drugs do not demonstrate this behavioral effect (Crawleyet al., “Absence of intrinsic antagonist actions of benzodiazepineantagonists on an exploratory model of anxiety in the mice”,Neuropharmacol. 23:531-537 (1984)).

Male N.I.H. Swiss-Webster mice (Harlan, Harlan, Indianapolis, Ind.)weighing 15-20 g were housed four per cage in polyethylene cages withsawdust bedding. The colony room was environmentally controlled (22° C.)with a 12 hr light/dark cycle (0600-1800 hr). Food and water wereavailable ad libitum, except during testing. The experiments were runfrom 0700-1500 hr and groups were counterbalanced for time of dayeffects. Mice were only administered drug or vehicle once.

The method used was a modification of methods previously described(Wieland et al., “Anxiolytic activity of progesterone metabolite5α-pregnan-3α-ol-20-one”, Br. Res. 565:263-268 (1991)). The apparatusincluded two 2-compartment automated test chambers (Model RXYZCM16,Omnitech Electronics, Columbus, Ohio). The open compartment wasconnected to the enclosed compartment via a 7.5×7.5 cm passageway. Theopen compartment was brightly lit using a 200 W incandescent light bulb.The experimental room was kept dark. Interruptions of the infrared beamsin either chamber were automatically recorded by being linked to acomputer through a Digiscan Analyzer (Omnitech Electronics) and the datawas analyzed using the Integrated Lab Animal Monitoring System (OmnitechElectronics). N.I.H. Swiss-Webster mice were administered vehicle ortest drug intraperitoneally (IP), 10 min later they were placed in thecenter of the lit compartment. The number of transitions between the litand dark chambers, total activity in the lit chamber and the time spentin the lit chamber were measured during a 10 min test period.

FIG. 8 shows the effects of 3α-OH-5α-pregnan-20-one and3α-OH-5β-pregnan-20-one in the light/dark transition test. Bothcompounds produced a significant dose-response curve in relation to thenumber of transitions between the dark box and the light box. Post-hoccomparisons showed that the number of the crossing for doses for both3α-OH-5α-pregnan-20-one and 3α-OH-5β-pregnan-20-one were significantlyincreased at doses tested from control (Dunnett's t-test).

In addition both 3α-OH-5α-pregnan-20-one and 3α-OH-5β-pregnan-20-oneproduced significant (p<0.01) increases in activity at 10 & 20 mg/kg ascompared to control groups (Dunnett's t-test). There were no significantdifferences between the two compounds at any dose tested.

b) Elevated Plus-Maze

The theoretical basis for the elevated plus-maze test is similar to thatof the light/dark transition test. As it was described previously(Pellow et al. “Validation of open:closed arm entries in an elevatedplus-maze as a measure of anxiety in the rat”, J. Neurosci. Meth.14:149-167 (1985)), the elevated plus-maze apparatus is designed toutilize the mice's natural aversion to open spaces. The apparatusconsists of two open-arms and two enclosed-arms. The elevated plus-mazetest allows for two measures of anxiety, the number of entries into theopen-arms and the time spent on the open-arms, both expressed as apercentage of the total number of entries and time spent in/on both theopen-arms and enclosed-arms.

Male N.I.H. Swiss-Webster mice (Harlan, Indianapolis, Ind.) weighing15-20 g were housed four per cage in polyethylene cages with sawdustbedding. The colony room was environmentally controlled (22° C.) with a12 hr light/dark cycle (0600-1800 hr). Food and water were available adlibitum, except during testing. The experiments were run from 0700-1500hr and groups were counterbalanced for time of day effects. Mice wereonly administered drug or vehicle once.

The method used was previously described (Lister, “The use of Plus-Mazeto measure anxiety in the mouse”, Psychopharmacol. 92:180-185 (1987)).The apparatus included two open arms perpendicular to two enclosed armselevated 50 cm from the floor. Each arm was 50 cm long and the walls ofthe enclosed arms were 40 cm tall. The maze was made completely of blackplexiglass. Incandescent 200 W light bulbs were above each of the openarms to produce a strong contrast between the open arms and the enclosedarms.

Ten minutes after an injection, the N.I.H. Swiss-Webster mice wereplaced in the center of the plus-maze facing an open arm. During the 5min test period, the number of entries onto the open arms and theenclosed arms, and the time spent in the open arms and enclosed armswere measured. All four paws had to be within an arm for the dependentvariable to be measured. Therefore, the time spent in the center of themaze is not counted, so the total time spent in the open arms and theenclosed arms may not equal 5 min. The effects of3α-OH-5α-pregnan-20-one and 3α-OH-5β-pregnan-20-one in the elevatedplus-maze test are shown in FIG. 9A. Both compounds demonstrate increaseproportion of entries into the open-arms across doses.3α-OH-5α-pregnan-20-one produced significant increase in entries at 20mg/kg (p≦0.05), whereas 3α-OH-5β-pregnan-20-one produced significantincreases in entries at 5 mg/kg (p≦0.05), 7.5 mg/kg (p≦0.01), and 10mg/kg (p≦0.01).

In addition, 3α-OH-5α-pregnan-20-one and 3α-OH-5β-pregnan-20-oneproduced dose-dependent increases in the time spent in the open-arms(FIG. 9B) 3α-OH-5α-pregnan-20-one produced significant increases in timespent on the open-arms at 10 mg/kg (p≦0.01), whereas3α-OH-5β-pregnan-20-one produced significant increases in time spent onthe open-arms at 7.5 mg/kg (p≦0.01) and 10 mg/kg (p≦0.01).

Table 8 shows the summary of anxiolytic activities of compounds of andused in the invention using the elevated plus-maze under the sameconditions described above.

TABLE 8 Anxiolytic Activity in Plus Maze in Mice Dose Name Route mg/kgVehicle % Control 3β-(4′-Acetylphenyl)ethynyl-3α-hydroxy-5α-pregnan- IP10 50% 175.7 20-one hpbcd3β-(4′-Acetylphenyl)ethynyl-3α-hydroxy-19-nor-5β- IP 10 50% 191pregnan-20-one hpbcd 3β-(4′-Hydroxybutyn-1′-yl)-3α-hydroxy-5β-pregnan-IP 10 50% 141 20-one hpbcd3β-(4′-Acetoxybutyn-1′-yl)-3α-hydroxy-5β-pregnan- IP 10 50% 159 20-onehpbcd 3α-Hydroxy-3(3-(3′-methoxy-1′-propynyl)-5β- IP 10 50% 134pregnan-20-one hpbcd 3β-[3-(2-Propynyloxy)propyn-1-yl]-3α-hydroxy-5β- IP10 50% 178 pregnan-20-one hpbcd3β-(4′-Hydroxybutyn-1′-yl)-3α-hydroxy-5α-pregnan- IP 10 50% 186 20-onehpbcd 3β-(4′-Biphenyl)ethynyl-3α-hydroxy-5β-pregnan-20- IP 10 dmso 111one 3β-(5-Acetylthien-2-yl)ethynyl-3α-Hydroxy-5β- IP 10 50% 83pregnan-20-one hpbcd 3β-(4′-Chlorophenyl)ethynyl-3α-hydroxy-5β-pregnan-IP 10 dmso 161 20-one 3β-(5′-Hydroxypentyn-1′-yl)-3α-hydroxy-5β-pregnan-IP 10 50% 137 20-one hpbcd3α-Hydroxy-3β-(thien-2-yl)ethynyl-5β-pregnan-20 IP 10 50% 148 one hpbcd3β-(4′-Acetylphenylethynyl)-3α,21-dihydroxy-5α- IP  1 50% 150pregnan-20-one hpbcd 3β-(4′-Acetylphenylethynyl)-3α,21-dihydroxy-5β- IP10 50% 115 pregnan-20-one hpbcd3β-(4′-Acetylphenylethynyl)-3α,21-dihydroxy-5α- PO 10 water 166pregnan-20-one 21-hemsuccinate salt3β-(4′-Acetylphenylethynyl)-3α,21-dihydroxy-5β- PO 10 water 248pregnan-20-one 21-hemsuccinate salt3β-(4′-hydroxy-1′-butynyl)-3α-hydroxy-5β-pregnan- IP 10 50% 11911,20-dione hpbcd 3β-(3′-Acetylphenyl)ethynyl-3α-hydroxy-5β-pregnan- IP10 50% 143 20-one hpbcd3β-(4′-Cyanophenyl)ethynyl-3α-hydroxy-5β-pregnan- LP 10 50% 169 20-onehpbcd 3β-(4′-Hydroxybutyn-1′-yl)-3α-hydroxy-5β-pregnan IP 10 water 14920-one 4′-hemisuccinate sodium salt3α-Hydroxy-3β-[3′-(1H-pyrazol-1-yl)-1′-propynyl]- IP 10 50% 1165β-pregnan-20-one hpbcd3α-Hydroxy-3β-(4′-methylphenyl)ethynyl-5β-pregnan- IP 50% 148 20-onehpbcd 3α-Hydroxy-3β-(6-oxo-1-heptynyl)-5β-pregnan-20- IP 10 50% 130.5one hpbcd 3α-Hydroxy-3β-(2′-pyridylethynyl)-5β-pregnan-20- IP 10 50% 123one hpbcd 3β-(4′-Cyano-1′-butynyl)-3α-hydroxy-5β-pregnan-20- IP 10 50%184 one hpbcd 3α-Hydroxy-3β-(3′-hydroxypropynyl)-5β-pregnan-20- IP 1050% 128 one hpbcd 3α-Hydroxy-3,3-[4′(R/S)-hydroxypentynyl]-5α- IP 10 50%102 pregnan-20-one hpbcd3α-Hydroxy-3,3-[3′(RS)-hydroxybutynyl]-5β-pregnan- IP 10 50% 137.720-one hpbcd 3β-(5′-Hydroxypentyn-1′-yl)-3α-hydroxy-5β-pregnan- IP 1050% 155 20-one 5′-hemisuccinate sodium salt hpbcd3β-(3′-Acetoxypropyn-1′-yl)-3α-hydroxy-5β-pregnan- IP 10 50% 109 20-onehpbcd 3α-Hydroxy-3(3-(2′-methoxyphenyl)ethynyl-5β- IP 10 dmso 152pregnan-20-one 3β-(6′-Acetoxyhexyn-1′-yl)-3α-hydroxy-5βpregnan- IP 1050% 206 20-one hpbcd 3α-Hydroxy-3β-[3′-1H-1,2,3-triazol-1-yl)-1′- IP 1050% 116 propynyl]-5(3-pregnan-20-one hpbcd3α-Hydroxy-3β-[3′-1H-1,2,4-triazol-1-yl)-1′- IP 10 50% 122propynyl]-5β-pregnan-20-one hpbcd SodiumS-(3α-hydroxy-5β-pregnan-20-on-21-yl) IP 10 50% 143 thiosulfate hpbcdSodium S-(3α-hydroxy-3β-methyl-5α-pregnan-20-on- IP 10 water 125 21-yl)thiosulfate Sodium S-[3α-hydroxy-3β-(4′-hydroxybutynyl)-5β- IP 10 water135 pregnan-20-on-21-yl] thiosulfate SodiumS-(3α-hydroxy-3β-methoxymethyl-5α- IP 10 water 126 pregnan-20-on-21-yl)thiosulfate Sodium S-(3α-hydroxy-3β-trifluoromethyl-19-nor-5β- IP 10 50%139 pregnan-20-on-21-yl)thiosulfate hpbcd3α-hydroxy-21-(2′H-1,2,3,4-tetrazol-2′-yl)-5β- IP 10 50% 117pregnan-20-one hpbcd 3α-Hydroxy-21-(2H-1,2,3-triazol-2-yl)-5β-pregnan-IP 10 50% 164 20-one hpbcd3α-Hydroxy-21-[1H-(4-methyl-5-carboxyl)imidazol-1 IP 10 50% 112yl)-5β-pregnan-20-one ethyl ester hpbcd3α-Hydroxy-21-(1′-imidazolyl)-5α-pregnan-20-one IP 10 50% 141 hpbcd3α-Hydroxy-21-(1′-imidazolyl)-5β-pregnan-20-one IP 10 50% 138 hpbcd3β-Ethynyl-3α-Hydroxy-21-(1′-imidazolyl)-5β- IP 10 50% 179pregnan-20-one hpbcd 3α-Hydroxy-21-(1H-3,5-dimethylpyrazolyl)-5β- IP 1050% 128 pregnan-20-one hpbcd 3α-Hydroxy-21-(1′-imidazolyl)-3β-methyl-5α-IP 10 50% 144 pregnan-20-one hpbcd3β-Ethynyl-3α-Hydroxy-21-(1′-pyrazolyl)-5β- IP 10 50% 180 pregnan-20-onehpbcd 3α-Hydroxy-21-(1′-pyrazolyl)-3β-methyl-5α-pregnan- IP 10 50% 15420-one hpbcd 3α-Hydroxy-21-(pyrazol-1-yl)-5β-pregnan-20-one IP 10 50%129 hpbcd 3α-Hydroxy-3β-methyl-21-(1′,2′,4′-triazolyl)-5α- IP 10 50% 153pregnan-20-one hpbcd3α-Hydroxy-21-(1′,2′,4′-triazol-1-yl)-5β-pregnan-20- IP 10 50% 135 onehpbcd 3α-Hydroxy-21-[1H-(2-methyl)imidazol-1-yl)-5β- IP 10 50% 96pregnan-20-one hpbcd

c) Geller-Seifter Conflict Test

This animal model of human anxiety utilizes a conditioned state ofconflict in rats to ascertain the anxiolytic properties of drugs. Ratsare conditioned to bar press for positive reinforcement under twoschedules of behavior (Geller and Seifter, “The effects of meprobamate,barbiturates, d-amphetamine and promazine on experimentally inducedconflict in the rat,” Psychopharmacologia 1:482-492 (1960)). The firstincludes bar pressing under a variable ratio schedule withoutpunishment. The second component is a fixed ratio schedule with each barpress resulting in a positive reinforcement and a punishment. Thepunished component produces a state of conflict within the animal. Theunpunished component allows for the observation of any responsedepressant effects a drug may possess. An anxiolytic response wouldincrease the punished responding without affecting the unpunishedresponding.

Male albino Sprague-Dawley rats (Charles River Labs, Wilmington, Mass.)weighing 250-300 g were used for conflict experiments and were kept on arestricted diet of Purina Lab Chow food pellets with water available atall times to maintain body weight at 85% of their free-feeding youngadult levels. Rats were housed individually under a 12-hour light-darkcycle with lights on from 0700-1900.

The anti-anxiety (punishment-lessening) and response depressant effectsof 3α-OH-5α-pregnan-20-one and 3α-OH-5β-pregnan-20-one were measured inrats by the conflict test of Geller and Seifter (1960). In this 63-mintest, hungry rats perform a lever-press response to obtain a sweetenedmilk reward. The reinforcement schedule consists of punishment andnonpunishment components, alternating approximately every 15 min. Ratswere trained in test chambers (Coulbourn instruments) with a levermounted in one wall, a small dipper that delivered the 0.1-mL milkreward (1 part Eagle condensed:milk 2 parts water), and a metal gridfloor through which the foot-shock punishment was administered. A DECPDP 11/73 minicomputer running SKED (State Systems) was used forprogramming and recording.

Rates initially learned to respond on a continuous reinforcementschedule and progressed rapidly to 30-sec, 1-min, and 2-min variableinterval (VI) schedules. On the continuous reinforcement schedule, ratsreceived milk reward following every lever press; on the VI schedules,milk rewards were available at infrequent and variable intervals,eventually at an average of once every 2 min. Four 3-min “conflict”periods were then introduced on the unpunished VI baseline; the firststarted after 3 min of VI performance and the others were alternatedbetween 12-min periods of VI responding. During conflict periods, whichwere signalled by the presentation of a light and a tone, the continuousreinforcement schedule was again in force and each lever press deliveredboth a milk reward and a brief (0.25 msec) foot-shock punishment. Shockintensity was 0.2 mA initially, and was increased daily in increments of0.02 mA in order to gradually suppress lever pressing to 5 responses orless per conflict period. This training took 4-6 weeks, after whichstable low rates of response were observed during conflict periods andstable high rates in the nonpunishment periods. Drug-induced increasesin the rate of punished responses were taken as an index of antianxietyactivity, while decreases in the rate of unpunished responses were takenas an index of response depression or sedation.

The effects of 3α-OH-5α-pregnan-20-one and 3α-OH-5β-pregnan-20-one inthe conflict test are summarized in FIG. 10. Both compounds producedlarge increases in the rate of punished responses, suggesting that bothwould be active as antianxiety agents. The peak effect of3α-OH-5β-pregnan-20-one was observed at 2 mg/kg and that of3α-OH-5α-pregnan-20-one at 4.4 mg/kg following subcutaneousadministration. (For statistical analysis and because of the smallnumber of tests at each dose, all tests with each compound were combinedfor comparison against vehicle control tests, using a t-test for relatedmeasures: for 3α-OH-5α-pregnan-20-one, p<0.02; for3α-OH-5β-pregnan-20-one, p<0.008).

Table 9 shows the summary of anxiolytic activities of compounds of andused in the invention using Geller-Seifter test under the experimentalconditions described above.

TABLE 9 Anxiolytic Activity in Geller/Seifter in Rats Geller/ SeifterDose (% of Compounds Route Vehicle (mg/kg) control)3α-Hydroxy-3β-methoxymethyl- IP 50% 10 958 5α-pregnan-20one hpbcd11α-N,N-Dimethylamino-3α- IP citrate 20 145hydroxy-3β-trifluoromethyl-5β- pregnan-20-one Sodium S-(3α-hydroxy-3β-PO water 32  4487.5 methoxymethyl-5α-pregnan-20- on-21-yl)thiosulfate3α-hydroxy-3β-ethoxymethyl- IP 50% 40 3743  5α-pregnan-20-one hpbcd

d) Vogel Test

The Vogel test is based on the development of a conflict between ahighly motivated behavior and an aversion. For this test, the strongmotivation is thirst. The animal is water deprived for 12-16 hrs toproduce the motivation to drink. During the training period the animalsare exposed to the testing environment so they become accustomed to thedrinking spout and minimize the fear of a novel environment. Followingtraining, the animals are allowed access to water for 2 hrs. During thistime, the animals will drink and eat their normal amount of water andfood, compensating for the deprivation time. However, this schedulestill produces a strong motivation to drink during the testing period.

Having been deprived of water for twelve to sixteen hours, an animal isplaced in a test cage where it is allowed to drink freely for fiveminutes. This period is used to habituate the animal to the environmentand the drinking spout. Following the training period, animals haveaccess to water and food in their home cage for 2 hrs. Food is availableat all times. Twenty-four hours later, drug is administered to theanimal intracerebroventricularly.

After an indicated delay started from the time of injection the animalis again placed in the test cage for ten minutes. A computer counts eachtime the animal licks, and after every twentieth lick administers a mildelectric stimulus across the tongue and/or feet. The electrical stimulusconsists of a 0.6 mA current with a duration of 100 msec. This procedureproduces a state of conflict for the animal that is reduced by theadministration of clinically used anxiolytic agents (i.e., Valium). Fordose-response curves, separate groups of animals are injected withincreasing doses of test drug and are tested at a predetermined time.

Data of representative compounds using these measurements are summarizedin Table 10.

TABLE 10 Anxiolytic Activity in Vogel Test in Rats Vogel Dose (% ofCompounds Route Vehicle (μg/kg) control) 3α,20α-Dihydroxy-2β- i.c.v.g-CD 10 169.0 isopropoxy-5α-pregnane 3α,20-Dihydroxy-20-methyl-5α-i.c.v. g-CD 20 179.0 pregnane 3α,20α-Dihydroxy-21-methyl- i.c.v. g-CD 10157.9 5α-pregnane 3α,20α(S)-Dihydroxy-5α- i.c.v. g-CD 10 193.0 pregnane3α-Hydroxy-5α-pregnan-20-one i.c.v. g-CD 10 431.13α,20α-Dihydroxy-5α-pregnane i.c.v g-CD 10 283.32β-Fluoro-3α,20α-dihydroxy- i.c.v. g-CD 20 264.9 5α-pregnane3α,20α-Dihydroxy-21-ethyl-5α- i.c.v. g-CD 20 225.8 pregnane3α,20α-Dihydroxy-5α-pregnane i.c.v. g-CD 20 267.3

Prodrugs

Anti-convulsant and anxiolytic activities of prodrugs of the basiccompounds 3α-hydroxy-5α-pregnan-20-one and3α,21-dihydroxy-5α-pregnan-20-one and their derivatives were assessed asusing the same procedures described above. Percent protection by severalprodrugs of 3α-hydroxy-5α-pregnan-20-one against metrazol-inducedseizures was plotted against time after administration of the compounds.(FIG. 11 and Table 11).

Modification of the basic compounds 3α-hydroxy-5α-pregnan-20-one and3α,21-dihydroxy-5α-pregnan-20-one at the 3αand 21 hydroxyls with variousesters maintains their biological activity and in some cases suchmodification increased the time of protection provided by the compound.Thus, the compounds of this invention can be modified to provideanti-convulsant and anxiolytic activities over a period of time, withvarying degrees of protection.

TABLE 11 Anti-Metrazol Activity of Prodrug Esters of3α-Hydroxy-5α-pregnane-20-one (3α-RCOO)-5α- pregnan-20-one) % Protection60 mg/kg 1 hr, R IP Methyl 25 Ethyl 75 Propyl 75 Butyl 33 2-Propyl 754-Heptyl 16 (4 hour) Cyclobutyl 17 Phenyl 33 4-Chlorophenyl 504-Methoxyphenyl 17 3-Pyridyl 50 (20 hour)3-(1-Methyl-1,4-dihydropyridinyl) 63 (20 hour)

In contrast to benzodiazepines, neuroactive steroids can also induceanesthesia. Their ability to induce anesthesia is thought to be due totheir ability to open the chloride ion channel in the absence of GABA,which is a property not possessed by benzodiazepines. Therefore,neurosteroids can act directly in the absence of GABA, at the receptor,and also “indirectly”, in the presence of GABA. This “indirect” actionis called “modulating” the receptor. (Lambert, et al., “Actions ofsynthetic and endogenous steroids on the GABA_(A) receptor,” TrendsPharmacology Science 8: 224-227 (1987).)

The compounds of and used in the invention can also be used foranesthetic indications at high doses. However, the preferred route ofadministration to induce anesthesia is intravenous (i.v.)administration. In animals, a drug's anesthetic properties is measuredby the drug's ability to produce a loss-of-righting reflex. Theloss-of-righting reflex is defined as the inability of an animal toright itself within 30 seconds when placed on its back. Mice wereadministered drug i.v. in the lateral tail vein. Followingadministration, mice were placed on their backs and observed forloss-of-righting reflex. Illustrative results are presented in Table 12.

TABLE 12 Anesthetic Activity of Neuroactive Steroids in Mice Loss-of-Dose Righting Compounds Route Vehicle (mg/kg) Reβex3α,21-Dihydroxy-3β-ethynyl- iv 20% 10 100 5β-pregnan-20-one cremophor3β-(Chloroethynyl)-3α- iv micronizing 20 100 hydroxy-5β-pregnan-20-onesolution 3β-Ethynyl-3α-hydroxy-5β- iv 10% hpbcd 30 100 pregnan-20-one3α-Hydroxy-3β-methyl-5α- iv micronizing 50 100 pregn-16-en-20-onesolution 3α-Hydroxy-5α-pregn-9-en- iv micronizing 10 100 20-one solution3α-Hydroxy-17(Z)- iv micronizing 5  75 methoxymethylene-19-nor- solution5α-androstane 3α-Hydroxy-3β-methyl-5β- iv micronizing 2.5 100pregnan-20-one solution 2β-Ethoxy-3α-hydroxy-5α- iv 20% 5 100pregnan-20-one cremophor 2β-Fluoro-3α-hydroxy-5α- iv micronizing 5 100pregnan-20-one solution 3α-Hydroxy-3β-methyl-21- iv micronizing 20 100methoxymethyl-5α-pregnan- solution 20-one

It is anticipated that prodrugs, with similar modifications as describedabove, of compounds of and used in the invention will have activity asprodrugs of 3α-hydroxy-5-reduced-pregnanes.

While the preferred embodiments have been described and illustrated,various substitutions and modifications may be made thereto withoutdeparting from the scope of the invention. Accordingly, it is to beunderstood that the present invention has been described by way ofillustration and not limitation.

What is claimed is:
 1. A method of treating or preventing stress oranxiety, treating or preventing mood disorders, alleviating orpreventing seizure activity, insomnia, premenstrual syndrome orpostnatal depression, or inducing anesthesia, comprising administeringto said animal subject in need of such treatment an effective amount ofa compound of Formula I:

wherein: R is hydrogen, halogen, lower alkoxy, alkyl, optionallysubstituted 1-alkynyl or substituted alkyl; R₁ is hydrogen, alkyl,alkenyl, aryl, aralkyl, alkynyl, optionally substituted aralkynyl,alkoxyalkyl, aminoalkyl, cyano, cyanoalkyl, thiocyanoalkyl, azidoalkyl,a substituted aralkynyl, arylalkyl, arylalkenyl, aryl, optionallysubstituted aralkylalkynyl, alkanoyloxyalkynyl, optionally substitutedheteroaryloxyalkynyl, oxoalkynyl or a ketal thereof, cyanoalkynyl,optionally substituted heteroarylalkynyl, hydroxyalkynyl, alkoxyalkynyl,aminoalkynyl, acylaminoalkynyl, mercaptoalkynyl, hydroxyalkynyl dioicacid hemi-ester or a salt thereof, or alkynyloxyalkynyl; R₂ is hydrogen,hydroxy, alkoxy, alkanoyloxy, carbalkoxyl, a keto group or amino group;R₃ is an optionally substituted heteroarylacetyl group; R₄ is hydrogenor methyl, R₅ is hydrogen; R₆ is hydrogen, alkanoyl, aminocarbonyl oralkoxycarbonyl; R₇ is hydrogen, halogen, hydroxy, alkoxy, alkanoyloxy,carbalkoxyl, a methylene group (together with R₃), or an alkoxymethylenegroup (together with R₃); R₈ is hydrogen or halogen; R₉ is hydrogen,halogen, alkyl, alkoxy, arylalkoxy or amino; and R₁₀ is hydrogen,halogen, alkyl, haloalkyl, hydroxy, alkoxy, alkanoyloxy, carbalkoxyl,cyano, thiocyano or mercapto; or a physiologically acceptable 3-ester,20-ester, 21-ester, 3,20-diester, or 3,21-diester thereof.
 2. The methodof claim 1, wherein said heteroarylacetyl group is imidazolylacetyl,pyrazolylacetyl, triazolylacetyl, tetrazolylacetyl, purinylacetyl,uracilacetyl or benzimidazolylacetyl.
 3. The method of claim 1, whereinsaid treating is alleviating or preventing stress or anxiety.
 4. Themethod of claim 1, wherein said treating is alleviating or preventingseizure activity.
 5. The method of claim 1, wherein said treating isalleviating or preventing insomnia.
 6. The method of claim 1, whereinsaid treating is alleviating or preventing premenstrual syndrome orpostnatal depression.
 7. The method of claim 1, wherein said treating isalleviating or preventing mood disorders.
 8. The method of claim 7,wherein said mood disorder is depression.
 9. A method of inducinganesthesia in an animal subject in need of said inducing, comprisingadministering to said animal subject an effective amount of a compoundof Formula I:

wherein: R is hydrogen, halogen, lower alkoxy, alkyl, optionallysubstituted 1-alkynyl or substituted alkyl; R₁ is hydrogen, alkyl,alkenyl, aryl, aralkyl, alkynyl, optionally substituted aralkynyl,alkoxyalkyl, aminoalkyl, cyano, cyanoalkyl, thiocyanoalkyl, azidoalkyl,a substituted aralkynyl, arylalkyl, arylalkenyl, aryl, optionallysubstituted aralkylalkynyl, alkanoyloxyalkynyl, optionally substitutedheteroarylalkynyl, oxoalkynyl or a ketal thereof, cyanoalkynyl,optionally substituted heteroarylalkynyl, hydroxyalkynyl, alkoxyalkynyl,aminoalkynyl, acylaminoalkynyl, mercaptoalkynyl, hydroxyalkynyl dioicacid hemi-ester or a salt thereof, or alkynyloxyalkynyl; R₂ is hydrogen,hydroxy, alkoxy, alkanoyloxy, carbalkoxyl, a keto group or amino group;R₃ is an optionally substituted heteroarylacetyl group; R₄ is hydrogenor methyl, R₅ is hydrogen; R₆ is hydrogen, alkanoyl, aminocarbonyl oralkoxycarbonyl; R₇ is hydrogen, halogen, hydroxy, alkoxy, alkanoyloxy,carbalkoxyl, a methylene group (together with R₃), or an alkoxymethylenegroup (together with R₃); R₈ is hydrogen or halogen; R₉ is hydrogen,halogen, alkyl, alkoxy, arylalkoxy or amino; and R₁₀ is hydrogen,halogen, alkyl, haloalkyl, hydroxy, alkoxy, alkanoyloxy, carbalkoxyl,cyano, thiocyano or mercapto; or a physiologically acceptable 3-ester,20-ester, 21-ester, 3,20-diester, or 3,21-diester thereof.
 10. A methodof modulating the GABA receptor-chloride ionophore complex in an animalsubject in need of said modulating through binding to the neurosteroidsite on said complex, comprising administering to said animal subject anamount, effective to modulate said complex, of a compound of Formula I:

wherein: R is hydrogen, halogen, lower alkoxy, alkyl, optionallysubstituted 1-alkynyl or substituted alkyl; R₁ is hydrogen, alkyl,alkenyl, aryl, aralkyl, alkynyl, optionally substituted aralkynyl,alkoxyalkyl, aminoalkyl, cyano, cyanoalkyl, thiocyanoalkyl, azidoalkyl,a substituted aralkynyl, arylalkyl, arylalkenyl, aryl, optionallysubstituted aralkylalkynyl, alkanoyloxyalkynyl, optionally substitutedheteroaryloxyalkynyl, oxoalkynyl or a ketal thereof, cyanoalkynyl,optionally substituted heteroarylalkynyl, hydroxyalkynyl, alkoxyalkynyl,aminoalkynyl, acylaminoalkynyl, mercaptoalkynyl, hydroxyalkynyl dioicacid hemi-ester or a salt thereof, or alkynyloxyalkynyl; R₂ is hydrogen,hydroxy, alkoxy, alkanoyloxy, carbalkoxyl, a keto group or amino group;R₃ is an optionally substituted heteroarylacetyl group; R₄ is hydrogenor methyl, R₅ is hydrogen; R₆ is hydrogen, alkanoyl, aminocarbonyl oralkoxycarbonyl; R₇ is hydrogen, halogen, hydroxy, alkoxy, alkanoyloxy,carbalkoxyl, a methylene group (together with R₃), or an alkoxymethylenegroup (together with R₃); R₈ is hydrogen or halogen; R₉ is hydrogen,halogen, alkyl, alkoxy, arylalkoxy or amino; and R₁₀ is hydrogen,halogen, alkyl, haloalkyl, hydroxy, alkoxy, alkanoyloxy, carbalkoxyl,cyano, thiocyano or mercapto; or a physiologically acceptable 3-ester,20-ester, 21-ester, 3,20-diester, or 3,21-diester thereof.
 11. A methodof treating anxiety and stress in an animal subject, comprisingadministering to said animal subject in need of such treatment aneffective amount of a compound of Formula I:

wherein: R is hydrogen, halogen, lower alkoxy, alkyl, optionallysubstituted 1-alkynyl or substituted alkyl; R₁ is hydrogen, alkyl,alkenyl, aryl, aralkyl, alkynyl, optionally substituted aralkynyl,alkoxyalkyl, aminoalkyl, cyano, cyanoalkyl, thiocyanoalkyl, azidoalkyl,a substituted aralkynyl, arylalkyl, arylalkenyl, aryl, optionallysubstituted aralkylalkynyl, alkanoyloxyalkynyl, optionally substitutedheteroaryloxyalkynyl, oxoalkynyl or a ketal thereof, cyanoalkynyl,optionally substituted heteroarylalkynyl, hydroxyalkynyl, alkoxyalkynyl,aminoalkynyl, acylaminoalkynyl, mercaptoalkynyl, hydroxyalkynyl dioicacid hemi-ester or a salt thereof, or alkynyloxyalkynyl; R₂ is hydrogen,hydroxy, alkoxy, alkanoyloxy, carbalkoxyl, a keto group or amino group;R₃ is an optionally substituted heteroarylacetyl group; R₄ is hydrogenor methyl, R₅ is hydrogen; R₆ is hydrogen, alkanoyl, aminocarbonyl oralkoxycarbonyl; R₇ is hydrogen, halogen, hydroxy, alkoxy, alkanoyloxy,carbalkoxyl, a methylene group (together with R₃), or an alkoxymethylenegroup (together with R₃); R₈ is hydrogen or halogen; R₉ is hydrogen,halogen, alkyl, alkoxy, arylalkoxy or amino; and R₁₀ is hydrogen,halogen, alkyl, haloalkyl, hydroxy, alkoxy, alkanoyloxy, carbalkoxyl,cyano, thiocyano or mercapto; or a physiologically acceptable 3-ester,20-ester, 21-ester, 3,20-diester, or 3,21-diester thereof.
 12. Themethod of claim 11, wherein said heteroarylacetyl group isimidazolylacetyl, pyrazolylacetyl, triazolylacetyl, tetrazolylacetyl,purinylacetyl, uracilacetyl or benzimidazolylacetyl.
 13. The method ofany one of claims 1, 9, 10 or 11, wherein said compound is apharmaceutically acceptable ester or diester of an acid selected fromthe group consisting of acetic, propionic, maleic, fumaric, ascorbic,pimelic, succinic, glutaric, bismethylenesalicylic, methanesulfonic,ethane-di-sulfonic, oxalic, tartaric, salicylic, citric, gluconic,itaconic, glycolic, p-aminobenzoic, aspartic, glutamic, γ-amino-butyric,α-(2-hydroxyethylamino)-propionic, glycine and other α-amino acids,phosphoric, sulfuric, glucuronic, and 1-methyl-1,4-dihydronicotinic. 14.The method of any one of claims 1, 9, or 11, wherein said effectiveamount is from about 1 mg to about 100 mg per dosage unit whenadministered intravenously and from about 100 mg to about 500 mg perdosage unit when administered non-intravenously.
 15. The method of anyone of claims 1, 9, 10 or 11 wherein R₃ is an optionally substitutedimidazolylacetyl group.
 16. The method of claim 15, wherein saidcompound is 3α-hydroxy-21-(1-imidazolyl)-5β-pregnan-20-one,3α-hydroxy-3β-(4-hydroxybutyn-1-yl)-21-(1-imidazolyl)-5β-pregnan-20-one,3α-hydroxy-3β-(4-hydroxybutyn-1-yl)-21-(1-imidazolyl)-5β-norpregnan-20-one,3α-hydroxy-21-[1H-(4-methyl-5-carboxyl)imidazol-1-yl)-5β-pregnan-20-oneethyl ester, 3α-hydroxy-21-(1-imidazolyl)-5α-pregnan-20-one,3β-ethynyl-3α-hydroxy-21-(1-imidazolyl)-5β-pregnan-20-one,3α-hydroxy-21-(1-imidazolyl)-3β-methyl-5α-pregnan-20-one,3α-hydroxy-21-[1H-(2-methyl)imidazol-1-yl)-5β-pregnan-20-one,3α-hydroxy-3β-trifluoromethyl-21-(1-imidazolyl)-5β-pregnan-20-one,3α-hydroxy-21-[1H-(2′-formyl)imidazol-1-yl)]-5β-pregnan-20-one, or3α-hydroxy-3β-trifluoromethyl-21-(1-imidazolyl)-5β-19-norpregnan-20-one.17. The method of any one of claims 1, 9, 10 or 11 wherein R₃ is anoptionally substituted pyrazolylacetyl group.
 18. The method of claim17, wherein said compound is3α-hydroxy-21-(pyrazol-1-yl)-5β-pregnan-20-one,3α-hydroxy-21-(pyrazol-1-yl)-5α-pregnan-20-one,3α-hydroxy-21-(1H-3,5-dimethylpyrazolyl)-5β-pregnan-20-one,3α-hydroxy-21-(1-pyrazolyl)-3β-methyl-5α-pregnan-20-one,3α-hydroxy-21-(pyrazol-1-yl)-3β-trifluoromethyl-5β-pregnan-20-one, or3α-hydroxy-21-(pyrazol-1-yl)-3β-trifluoromethyl-5β-19-nor-pregnan-20-one.19. The method of any one of claims 1, 9, 10, or 11 wherein R₃ is anoptionally substituted triazolylacetyl group.
 20. The method of claim19, wherein said compound is3α-hydroxy-3β-methyl-21-(1,2,4-triazolyl)-5α-pregnan-20-one,3α-hydroxy-3β-(4-hydroxybutyn-1-yl)-21-(1,2,3-triazol-2-yl)-5β-19-norpregnan-20-one,3α-hydroxy-21-(2H-1,2,3-triazol-2-yl)-5β-pregnan-20-one,3α-hydroxy-3β-methyl-21-(2′H-1,2,3-triazol-2-yl)-5α-pregnan-20-one,3β-ethynyl-3α-hydroxy-21-(1,2,4-triazolyl)-5β-pregnan-20-one,3α-hydroxy-3β-methyl-21-(1,2,3-triazol-1-yl)-5α-pregnan-20-one,3α-hydroxy-21-(1,2,4-triazol-1-yl)-5β-pregnan-20-one,3α-hydroxy-3β-(4-hydroxybutyn-1-yl)-21-(1,2,4-triazol-1-yl)-5β-pregnan-20-one,3α-hydroxy-3β-(4-hydroxybutyn-1-yl)-21-(1,2,3-triazol-1-yl)-5β-pregnan-20-one,3α-hydroxy-3β-(4-hydroxybutyn-1-yl)-21-(1H-1,2,3-triazol-1-yl)-5β-19-norpregnan-20-one,3α-hydroxy-3β-trifluoromethyl-21-(1H-1,2,3-triazol-1-yl)-5β-19-norpregnan-20-one,or3α-hydroxy-3β-trifluoromethyl-21-(1H-1,2,3-triazol-1-yl)-5β-19-pregnan-20-one.21. The method of any one of claims 1, 9, 10, or 11 wherein R₃ is anoptionally substituted tetrazolylacetyl group.
 22. The method of claim21, wherein said compound is3α-hydroxy-21-(2H-1,2,3,4-tetrazol-2-yl)-5β-pregnan-20-one,3α-hydroxy-21-(1H-1,2,3,4-tetrazol-1-yl)-5β-pregnan-20-one, or3α-hydroxy-3β-(4-hydroxybutyn-1-yl)-21-(tetrazol-1-yl)-5β-pregnan-20-one.